WO2003054166A2

WO2003054166A2 - Nucleotide polymorphisms associated with osteoarthritis

Info

Publication number: WO2003054166A2
Application number: PCT/US2002/041225
Authority: WO
Inventors: Karen Anne Jones; Alan Schafer
Original assignee: Incyte Genomics, Inc.
Priority date: 2001-12-20
Filing date: 2002-12-19
Publication date: 2003-07-03
Also published as: AU2002366713A8; AU2002366713A1; WO2003054166A3

Abstract

The invention relates to novel polynucleotides associated with human disease, and in particular to osteoarthritis. The invention further relates to polymorphic polynucleotides associated with osteoarthritis. The invention provides methods of determining if a particular polymorphism predisposes an individual to or is associated with the development of osteoarthritis. The invention also provides methods of detecting the presence of one or more polymorphism as an indicator of osteoarthritis, and provides for use of novel polynucleotides of the invention in the development of drugs and in disease treatment.

Description

NUCLEOTIDE POLYMORPHISMS ASSOCIATED TTH OSTEOARTHTRTTS

TECHNICAL FIELD The invention relates in general to polymorphisms in genes associated with osteoarthritis and bone remodeling and methods of identifying individuals having a gene contai-oing a polymorphism associated with osteoarthritis. The invention also relates to a method of detecting an increases susceptibility to a disease in an individual resulting from the presence of a polymorphism or mutation in the gene coding sequence of a osteoarthritis and bone remodeling associated gene.

BACKGROUND OF THE INVENTION

Single nucleotide substitutions and small unique insertions and deletions are the most frequent form of DNA polymorphism and disease-causing mutation in the human genome. These DNA sequence variations, called single nucleotide polymorphisms (SNPs), have gained popularity and have been proposed as the genetic markers of choice for the study of complex genetic traits (Collins et al. 1997 Science 278: 1580- 1581; Risch and Merkangas 1996 Science 273: 1516-1517). Despite the fact that on average approximately one nucleotide position in every 1000 bases along the human chromosome is estimated to differ between any two copies of the chromosome (Cooper et al. 1985 Human Genetics 69: 201-205; Kwok et al. 1996 Genomics 31: 123-126) developing SNP markers is not easy. It has been suggested that association studies (such as linkage equilibrium studies) with a set of single nucleotide polymorphism (SNP) markers evenly spaced across the genome at approximately 100 KB intervals would provide the necessary power to detect the small effects of each gene involved in a complex trait (Hauser et al. 1996 Genetic Epidemiology 13: 117-137 in Kwok and Chen 1998 Genetic Engineering 20: 125-134, Plenum Press, New York). Alternatively, one can take a candidate gene approach in performing association studies with the use of a set of gene-associated SNP markers to detect these genetic factors (ibid.).

Nucleotide sequence mutations which occur in a gene or gene family, where the gene or gene family is associated with a given disease, may be the basis for susceptibility to or development of the disease. Arthritis means ''inflammation of a joint" and encompasses more man a hundred diseases.

They can affect the joints and other connective tissues such as muscles, tendons, ligaments and protective coverings of internal organs. The major arthritis diseases are as follows:

1. osteoarthritis - non-iiiflammatory degenerative joint disease characterised by splitting and fragmentation of the articular cartilage, hypertrophy of the bone and changes in the synovial membrane.

2. rheumatoid arthritis - chronic systemic, relapsing disease primarily of the joints which is marked by inflammatory changes in the synovial membranes and adjacent structures. 3. ankylosing spondylitis - inflammatory disease that affects the joints of the lower back which may lead to fusion of the spine

4. gout - caused by formation of uric acid crystals in the joint, leading to inflammation and severe pain.

Osteoarthritis is the most common type of arthritis. It differs from rheumatoid arthritis in that it is primarily a degeneration of the joint tissue that may be accompanied by an inflammatory reaction (Figure 1). Rheumatoid arthritis is an inflammatory disease first and foremost and inflammation of the synovium is the focal point of the disease.

The initiation and progression of osteoarthritis involves multiple pathogenic mechanisms. An imbalance of chondrocyte-controlled anabolic and catabolic processes results in a progressive degradation of the components of the extracellular matrix of the articular cartilage, associated with , secondary inflammatory factors. The primary cause of this is unknown but possibly involves ,a . > ,.. ' deficiency of cellular; response to normal tissue demand or insufficient cellular response to , ... ' ^•■< . }• - supernormal demand from mechanical loading or injury. The subsequent repair response could induce .. elevated levels of anabolic molecules, leading to remodelling of the bone and production of osteophytes (bone outgrowths) characteristic of the disease process. • ;^{■ • • "}' ^{•• ■}

Prevalence and social cost of osteoarthritis.

With approximately 40 million Americans affected by arthritis and other inflammatory diseases, the cost to the healthcare system is significant. Of tliese 40 million people, 21 million have osteoarthritis and 2.1 million have rheumatoid arthritis. Osteoarthritis is the most common chronic condition and cause of inactivity in patients older than 65. The disease occurs usually at the begirining of the fifth decade of life, with increasing prevalence and incidence with advancing age (Table 2). The prevalence of arthritis is expected to increase by 57% by the year 2020. In the same time period, arthritis-causing activity limitation will increase 66% to 11.6 million people (Lawrence et al 1998). The primary impact of arthritis in the elderly is decreased physical functioning. This can be due to other health-related problems, such as weight gain, cardiovascular disease, GI distress related to treatment, increased psychological distress, decreased social functioning, increased work disability, and increased healthcare utilization. The current OA treatment, NSAJDs are responsible for the highest number of hospitalisations of any drug category and cause a significant number of internal gastrointestinal bleeding in the elderly population.

The cost of arthritis in the US (including rheumatoid arthritis, osteoarthritis and all other rheumatic conditions) was shown to be $64.8 billion in 1992. Of this, direct costs were an estimated $15.2 billion and indirect costs $49.6 billion (Yelin and Callahan 1995). A 1997 study showed the cost of care for osteoarthritis as $543 per patient per year (Lanes et al 1997). The largest component was hospital care, mostly due to admissions for hip or knee replacement. The cost to the healthcare provider is very high due to the prevalence of the illness. Unmet medical needs for OA

Current treatment options for osteoarthritis focus on symptom relief whereas truly disease- modifying agents or methods are lacking. Thus, the basic therapy includes common analgesics, nonsteroidal anti-inflammatory drugs, physical therapy, walking aids, and eventually in severe cases, joint replacement surgery. Perhaps because of the difficulties involved in measuring disease progression existing medications do not address the need to prevent further cartilage degradation.

i » i To'develop such drugs the following should be in place:

Compounds that target appropriate biochemical pathways (e.g. Merk's MMP-3 antagonist) - Clinical studies must be able to measure disease progression in a cost-effective and safe fashion. This could be either an imaging teclinique or a biomarker that closely correlates with disease progression.

Disease progression should be detectable within a reasonable time scale (for example, anti- inflammatory clinical studies use the WOMAC pain scale for a period of 6 weeks to measure improvement due to medication).

The efficacy of the new drug under development should be observable (using either the imaging or biomarker method of assessment) in a sample size comparable to that of other clinical trials.

How can genetics help? Genetic studies have the potential to detect:

Novel drug targets in the appropriate pathways. Individuals with fast progressing osteoarthritis. This would allow a pharmaceutical company to prove efficacy in a relatively small sample size and in a reasonable period of time, thus cutting costs.

Reduce variation from biomarker or imaging patterns. For example, let's assume the following response to medication. Although there is a clear patterns of response to medication, it is not statistically significant because of the large amount of variation in disease progression. Lets now assume that there exists a genetic marker that is able to stratify the measurement of disease progression in this hypothetical study. The variance of the marker of disease progression associated with each genotype is smaller than the overall variance. This can be seen as analogous to stratifying a relevant clinical measure in a study (e.g. lipid levels) by gender or by age group. By pooling together both genders or both age groups the variance is larger. If we were now to stratify the results of the previous hypothetical study by genotype we might observe that the therapeutic efficacy is now statistically significant. By stratifying according to genotype it could then be possible to detect statistically significant efficacy in both groups, while meeting the cost and time needs of the entity developing the drug.

Genetic study of osteoarthritis.

Evidence for genetic predisposition to OA. ι • .

The nature of the genetic influence in osteoarthritis may involve either a structural defect (that ■ is, collagen), alterations in cartilage or bone metabolism, or a genetic influence on a known risk factor for osteoarthritis such as obesity. Twin studies have show that between 39% and 65% of osteoarthritis in the general population can be attributed to genetic factors (MacGregor and Spector, 1999). Linkage analyses (i.e., common inheritance of affected individuals in the same family) have identified a higher risk ratio for relatives of affected individuals compared to the general population. The power to detect disease-susceptibility loci through linkage analysis using pairs of affected relatives depends on 1_R, the risk ratio for type R relatives compared with population prevalence (Risch 1990). Kellgren et al. (1963) compared expected and observed incidence of osteoarthritis in first-degree relatives of probands with multiple osteoarthritis. Based on their results we have estimated 1_R for nodal and non-nodal osteoarthritis.

Modal (presence of Heberdeen's nodes) 4.5 Non-nodal 4.75 For comparison, concordance for type 2 diabetes ranges between 2-3, and between 4.5 and 5.5 for rheumatoid ailhritis. These figures indicate a high genetic component to OA If, however, non- nodal and nodal types of OA are mixed together 1_R drops to ~ 2.0 highlighting the importance of careful clinical characterization for genetic studies. Although it is known that there is a genetic component involved in the etiology of osteoarthritis there is also a need in the art for an improved understanding of the genetic causes of osteoarthritis.

There is also a need in the art for identification of the genes associated with osteoarthritis, and identification of sequence variations in these genes that are associated with osteoarthritis and bone remodeling. The identification of disease related sequence variations in osteoarthritis and bone remodeling associated genes will allow for the development of improved methods of screening for osteoarthritis. These improved screening protocols may be used to identify individuals at high risk for osteoarthritis and in need of preventative treatments.

The identification of disease related sequence variations in osteoarthritis associated genes may facilitate the design of treatment protocols and the identification and design of compounds useful for treatment of osteoarthritis and bone remodeling.

OBJECTS AND SUMMARY OF THE INVENTION

^{■ ■}■ ■^• > ^• An object of the present invention is to, provide candidate genes associated with osteoarthritis . and bone remodeling. ^{• :} . , v ^■ ... . ^{' •"}:. It is another object of the present invention to provide a variant nucleotide in a candidate'gene associated with osteoarthritis and bone remodeling.

Another object of the present invention is to provide methods of detecting variant nucleotides in a gene in individuals at risk for osteoarthritis.

Another object of the present invention is to provide methods of deternώώng if a variant nucleotide is associated with a predisposition to osteoarthritis.

Another object of the present invention is to provide candidate genes associated with the osteoarthritis and bone remodeling.

The invention further comprises isolated polynucleotides which contain the single nucleotide polymorphisms selected from the Sequence Listing, or its perfect complement. The invention further comprises an isolated polynucleotide segment of between 10 and 100 bases of which 10 contiguous bases including a polymorphic site are from a sequence selected from the Sequence Listing, or its perfect complement.

The invention further comprises a probe or target sequence used for genotyping where the probe or target sequence has at least 10 contiguous bases containing a polymorphic site identified and from a sequence selected from the Sequence Listing, or its perfect complement.

The invention further comprises a method for deteiTnining a base occupying a polymorphic site in a nucleic acid comprising obtaining the nucleic acid in a sample from an individual or plurality of individuals and determining a base occupying a polymorphic site in a sequence selected from the group consisting of the Sequence Listing and their perfect complements which occurs in the sample nucleic acid.

DESCRIPTION OF THE COMPACT DISK-RECORDABLES (CD-R) CD-R (Copy l)contains the Sequence Listing formatted in plain ASCII text and Tables 1 and

2. CD-R (Copy 1) is labeled with Identification No. GX-0022P-1.

CD-R (Copy 2) is an exact copy of CD-R (Copy 1). CD-R (Copy 2) is labeled with Identification No. GX-0022-1 P (Copy 2).

CD-R (Copy 3) contains the Computer Readable Form of the Sequence Listing in compliance with 37 C.F.R. §1.821(e), and specified by 37 C.F.R. §1.824. CD-R (Copy 3) is labeled with Identification No. GX-0022-1 P (Copy 3).

The material on CD-R 1, 2 and 3 is incorporated by reference into the specification.

BRIEF DESCRIPTION OF THE TABLES AND DRAWINGS These and other features, aspects, and advantages of the present invention will become better understood with regard to the following description, appended claims, and accompanying tables drawings where:

Table 1 presents the genomic or cDNA structure of osteoarthritis candidate gene sequences and the identity and position of polymorphisms which are the subject of the invention. This table has the form wherein: a. The DNA change given for an allele is not strand specific; it can be on either strand of the DNA molecule. b. Single Nucleotide Polymorphisms can be recorded as IUPAC ambiguity symbols, as follows: M A or C

R A or G

S C or G

K G or T W A or T Y C or T c. Other allele types, such as insertions and deletions, are given in the form: ACA>AA or AA>ACA and in such cases the coordinates of the allele include the two invariant flanking bases. d. DNA sequence names are of the form: XX:III I II[_VV], where XX gives the database of origin, as follows:

EM EMBL

FN Incyte FL sequence read GB GenBank

IN Incyte proprietary sequence LG LifeSeq Gold gene template

1111111 gives the sequence ID or accession number for the sequence. In most cases if it is an accession number it will be followed by _VV where VV is the sequence version in the EMBL or GenBank database. e. The overall structure. of a record in the patent, structure is described as follows. Items in {braces} indicate a field that is filled in. Items in [square brackets] may or may not be present. These entries define a larger virtual sequence;- a "link" - composed of real database subsequences. AUeles are annotated onto real sequences, and genomic structure onto the link. (Locus ID}

[Full name : {full name}] Link : {link name}

Subsequence {name} {link start position} {link stop position} {SEQ ID NO} [...]

CDS {name} {SEQ ID NO} exon/ORF {link start position} {link stop position}

[•••] Allele {seq name} {SEQ ID NO} {seq start} {seq stop} {dna change} source {original SNP data source} {SNP id in that source}

[•••] consequence {CDS name} {CDS SEQ ID NO} {class} [{peptide pos} {peptide change}]

[•••] f. Sources. SNPs may have been noted in one of several sources: dbSNP The NCBI public dbSNP databank isSNP In silico SNPs from LifeSeq sequence assembly. wetSNP Alleles determined by SSCP. Alleles which have a wetSNP entry are experimentally verified. Alleles which are isSNP and/or dbSNP only are predictions by computer software of where these SNPs map to, and are *not* experimentally verified. g. Consequences

The classes of consequence are as follows:

Silent The allele does not cause a peptide change Missense The allele causes an amino acid substitution Framesbift The allele causes a frame shift in the CDS

Intron The allele lies wholly within an intron . .. 5' The:allele lies 5'Of the,CDS ^■. . - ^•■ . ■

3' The- allele lies 3 ' of the CDS

: > ^■ Unknown The consequence is undefined - for example the allele straddles an intron/exon boundary. -

Silent and Missense consequences also supply details of the amino acid position of the change, and prediction of what the affected amino acid is, and what it is substituted to. There may be multiple consequence lines if the locus contains multiple CDS forms, h. Sequence and exon positions Sequence coordinates are always given on the forward strand of the link. Therefore, if a sequence or exon is actually on the reverse strand of the link, its start position will be larger than its stop position. i. Exon order in CDS definitions

The exons are given in 5 'to 3' order. Consequently, reverse strand CDS start from high coordinate numbers downwards, j. Link object types Loci may have more than one link object, composed of different DNA sequences. Typically there might be one genomic and one cDNA link object. Table 2 presents the population frequency of polymorphisms in the candidate genes and summarizes various information from Table 2 relating to. the polymorphism.

Figure 1 illustrates the cDNA structure of the locus and relative positions of identified SNPs for megakaryocyte stimulating factor (MSF). Figure 2 illustrates the genomic structure of the locus, exons composing multiple CDS, and relative positions of identified SNPs for megakaryocyte stimulating factor (MSF).

The figures show (from left to right) the real sequences making up the linked genomic structure for the locus, a scale in link coordinates (negative numbers would indicate a view of the reverse strand), one or more CDSs representing the positions of exons, horizontal bars representing the positions of identified SNPs (alleles) from the various sources, and shaded boxes showing regions targeted for screening by SSCP.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

Before the present compositions and methods are described, it is understood that embodiments of the invention are not limited to the particular machines, instruments, materials, and methods described, as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the invention.

As used herein and in the appended claims, the singular forms "a," "an," and "the" include plural reference unless the context clearly dictates otherwise. Thus, for example, a reference to "a nucleic acid probe" includes a plurality of such nucleic acid probes, and a reference to "a gene" is a reference to one or more genes and equivalents thereof known to those skilled in the art, and so forth. Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any machines, materials, and methods similar or equivalent to those described herein can be used to practice or test the present invention, the preferred machines, materials and methods are now described. All publications mentioned herein are cited for the purpose of describing and disclosing the cell lines, protocols, reagents and vectors which are reported in the publications and which might be used in connection with various embodiments of the invention. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention. Definitions As used herein, "polymorphism" refers to a nucleotide alteration that either predisposes an individual to a disease or is not associated with a disease, which occurs as a result of a substitution, insertion or deletion.

More particularly, a "polymorphism" or "polymorphic variation" may be a nucleic acid sequence variation, as compared to the naturally occurring sequence, resulting from either a nucleotide deletion, an insertion or addition, or a substitution, which is present at a frequency of greater than 1% in a population.

As used herein, "neutral polymorphism" refers to a polymorphism which is present at a frequency of greater than 1% in a population, which does not alter gene function or phenotype, and thus is not associated with a predisposition to or development of a disease.

As used herein "polynucleotide sequence" refers to a sense or antisense nucleic acid sequence comprising RNA, cDNA, genomic DNA, synthetic forms and mixed polymers, that maybe chemically or biochemically modified or may contain non-natural or derivatized nucleotide bases. As used herein "mutation" refers to a variation in the nucleotide sequence of a gene or regulatory sequence as compared to the naturally occurring or normal nucleotide sequence. A mutation may result from the deletion, insertion or substitution of more than one nucleotide (e.g., 2, 3 , 4, or more nucleotides) or a single nucleotide change such as a deletion, insertion or substitution. The term "mutation" also encompasses chromosomal rearrangements.

As used herein, "nucleic acid probe" refers to an oligonucleotide, nucleotide or polynucleotide, and fragments and portions thereof, and to DNA or RNA of genomic or synthetic origin which may be single- or double- stranded, which represents the sense or antisense strand. Both terms "nucleic acid probe" and "DNA fragment" refer to a length of polynucleotide, for example, as small as 5 nucleotides, 10, 20, 25, 40, 50, 75, 100, 250, 400, 500 and 1 kb, and as large as 5-lOkb.

As used herein, "alteration" refers to a change in either a nucleotide or amino acid sequence, as compared to the naturally occurring sequence, resulting from a deletion, an insertion or addition, or a substitution.

As used herein, "deletion" refers to a change in either nucleotide or amino acid sequence wherein one or more nucleotides or amino acid residues, respectively, are absent.

As used herein, "insertion" or "addition" refers to a change in either nucleotide or amino acid sequence wherein one or more nucleotides or amino acid residues, respectively, have been added. As used herein, "substitution" refers to a replacement of one or more nucleotides or a ino acids by different nucleotides or amino acid residues, respectively.

As used herein, "specifically hybridizable" refers to a nucleic acid or fragment thereof that hybridizes to another nucleic acid (or a complementary strand thereof) due to the presence of a region that is at least approximately 90% homologous, preferably at least approximately 90-95% homologous, and more preferably approximately 98-100% homologous, as are polynucleotides that hybridize to a partner under stringent hybridization conditions. "Stringent" hybridization conditions are defined hereinbelow for various hybridization protocols. A probe that is specifically hybridizable to a given sequence can be used to detect a 1 bp out of 10 bp (10%) or a 1 bp out of 2O bp (5 %) difference between nucleic acid sequences and is therefore useful for discriminating between a wild type and a mutant form of a gene of interest.

As used herein, "amino acid sequence" refers to the sequential array of amino acids that have been joined by peptide bonds between the carboxylic acid group of one amino acid and the amino group of the adjacent amino acid to form long linear polymers comprising proteins.

As used herein, "amino acid" refers to protein subunit molecules that contain a carboxylic acid group, and an amino group, both linked to a single carbon atom.

A polypeptide is said to be "encoded" by a polynucleotide if the polynucleotide, either in its native state or in a recombinant form can be transcribed and/or translated to produce the mRNA for and/or the polypeptide or a fragment thereof.

As used herein, "gene " refers to a region of DNA which includes a portion which can be , transcribed into RNA, and which may contain an open reading frame, or coding region (also referred . to as an' exon) which encodes a protein, a non-coding region (also referred to as an intron), and a specific regulatory region comprising the DNA regulatory elements which control expression of the transcribed region.

As used herein, "coding region" refers to a region of DNA which encodes a protein, also known as an exon.

As used herein, "non-coding region" refers to a region of DNA which does not encode a protein coding region, also known as an intron, and is not included in the RNA molecule that is synthesized from a particular gene.

As used herein, "regulatory region" refers to DNA sequences which are located either 5' of the transcription start site, 3' or the transcription termination site, within an intron or exon, capable of ensuring that the gene is transcribed at the proper time and in the appropriate cell type. As used herein, "consensus DNA sequence" or "wild-type DNA sequence" refers to a sequence wherein every position represents the nucleotide that occurs with the highest frequency when many actual sequences are compared. As used herein, "consensus DNA sequence" or "wild- type DNA sequence" also refers to the normal, naturally occurring DNA sequence. As used herein, a given sequence (or mutation or polymorphism) "associated with" osteoarthritis refers to a nucleic acid sequence that increases susceptibility to the disease, predisposes an individual to the disease or contributes to the disease, wherein the nucleic acid sequence is present at a higher frequency (at least 5%, preferably 10%, more preferably 25% higher) in individuals with the disease as compared to individuals who do not have the disease.

As used herein, a sequence "not associated with" osteoarthritis refers to a nucleic acid sequence that does not increase susceptibility to the disease, predispose an individual to the disease or contribute to the disease, wherein the nucleic acid sequence is not present at a higher frequency in individuals with the disease, and thus is present at a frequency about equal to its frequency in individuals who do not have the disease.

As used herein, "amplifying" refers to producing additional copies of a nucleic acid sequence, preferably by the method of polymerase chain reaction (Mulhs and Faloona, 1987, Methods Enzymol. 155: 335).

As used herein, "oligonucleotide primers" refer to single stranded DNA or RNA molecules that are hybridizable to a nucleic acid template and prime enzymatic syntliesis of a second nucleic acid strand. Oligonucleotide primers useful according to the invention are between 5 to 100 nucleotides in length, preferably 20-60 nucleotides in length, and more preferably ^'20-40 nucleotides in length.

As used herein, "sequencing" refers to deterniining the precise nucleotide composition or sequence of a nucleic acid region by methods well known in the art (see Ausubel et al., supra and Sambrook et al, supra).

As used herein, "comparing" a sequence refers to determining if the nucleotides at one or more positions in a particular region of a nucleic acid fragment are identical for any two or more sequences. According to the invention, sequence comparisons can be performed by using computer program analysis as described below in Section F entitled "Identification and Characterization of Polymorphisms".

As used herein, "sequence differences" or "sequence variations" refer to nucleotide changes, at one or more positions between any two or more sequences being compared.

As used herein, "determining the presence of polymorphic variations" refers to using methods well known in the art to identify a nucleotide, at one or more positions within a particular nucleic acid region, that is distinct from the nucleotide present in the naturally occurring, wild-type or consensus sequence, resulting from either a nucleotide deletion, an insertion or addition, or a substitution.

As used herein, "determixiing the absence of polymorphic variations" refers to using methods well known in the art to determine that the nucleotides present at every position analyzed in a particular nucleic acid region are identical to the nucleotides present in the naturally occurring, wild- type or consensus sequence.

As used herein, "genotyping" refers to determining the composition of the genetic material that is inherited by an organism from its parents. As used herein, "biological sample" refers to a tissue or fluid sample containing a polynucleotide or polypeptide of interest, and isolated from an individual including but not limited to plasma, serum, spinal fluid, lymph fluid, urine, stool, external secretions of the skin, respiratory, intestinal and genitoruinary tracts, sahva, blood cells, tumors, organs, tissue and samples of in vitro cell culture constituents. As used herein, "amplimers" refer to a specific fragment of DNA generated by PCR that is at least 30 bp in length and is preferably between 50 and lOObp in length, and is more preferably between 150-300bp in length, with a melting temperature in the range of approximately 60-62°C.

As used herein, "phenotype" refers to the biological appearances of an organism or a tissue derived from an organism, wherein biological appearances include chemical, structural and behavioral attributes, and excludes genetic constitution.

As used herein, "genotype" refers to the genetic material that is inherited by an organism from its parents.

As used herein, "genetic susceptibility to osteoarthritis" refers to an increased risk of developing osteoarthritis resulting from specific DNA differences relative to non-susceptible individuals. Preferably an individual who is genetically susceptible to osteoarthritis has a 5-100%, and more preferably a 25-50% greater chance of developing osteoarthritis, as compared to non- susceptible individuals.

As used herein, "diagnostic" refers to the practice of identifying a disease from the signs and symptoms of an individual including the DNA sequences of genes that are associated with an increased susceptibihty to the disease. "Diagnostic" also refers to the practice of stratifying patient populations based on the efficacy or toxicity of a composition, and the predictive placement of an individual in a response strata based on stata-associated parameters.

As used herein, "prognosis" refers to the possibility of recovering from a particular disease or condition, and also refers to risk assessment of developing a particular disease or condition.

THE INVENTION

Various embodiments of the invention include polynucleotides and polymorphic polynucleotides associated with a given human disease, for example, with osteoarthritis. The invention also provides a gene sequence containing one or more polymorphic nucleotides associated with a predisposition to or the development of a given human disease such as osteoarthritis. The invention also relates to polypeptides encoded by the polynucleotides or the polymorpWsm-containing gene. The invention also provides methods of detecting a polymorphism according to the invention in individuals at risk for osteoarthritis, and for determining if a given polymorphism is associated with a predisposition to the disease. The invention also discloses polymorphism(s) that are either associated with or are not associated with (i.e., are neutral) osteoartliritis. A polymorphism in a given gene can be utihzed in various diagnostic and therapeutic methods and procedures, for example, in nucleic acid and peptide diagnosis, drug screening and design, and in gene and peptide therapy. A polymorphism associated with a given gene can be utihzed in various gene expression systems and assays designed to analyze gene regulation and expression.

A. Design and Synthesis of Oligonucleotide Primers

According to the present invention, ohgonucleotide primers are disclosed that are useful for deterrrήning the sequence of a particular allele of a gene. The invention also discloses ohgonucleotide primers designed to amplify a region of a gene that is known to contain a polymorphism.. The invention also discloses ohgonucleotide primers designed to anneal specifically to a particular allele of a gene. ,\

Ohgonucleotide primers useful according to the invention are single-stranded DNA or RNA molecules that are hybridizable to a nucleic acid template and prime enzymatic synthesis of a second ; nucleic acid strand. The primer is complementary to a portion of a target molecule present in a pool of nucleic acid molecules. It is contemplated that ohgonucleotide primers according to the invention are prepared by synthetic methods, either chemical or enzymatic. Alternatively, such a molecule or a fragment thereof is naturaUy-occurring, and is isolated from its natural source or purchased from a commercial supplier. Ohgonucleotide primers are 5 to 100 nucleotides in length, ideally from 20 to 40 nucleotides, although oligonucleotides of different length are of use.

Pairs of single-stranded DNA primers can be annealed to sequences within or surrounding a gene on chromosome Y in order to prime amplifying DNA synthesis of a region of a gene. A complete set of gene primers will allow synthesis of ah of the nucleotides of the coding sequences, e.g., the exons, introns and control regions. Preferably, the set of primers will also allow synthesis of both intron and exon sequences.

Ahele-specific primers are also useful, according to the invention. Such primers will anneal only to a particular-mutant allele (e.g. alleles containing a polymorphism), and thus will only amplify a product if the template also contains the polymorphism. Allele specific primers that anneal only to a wild type gene sequence are also useful according to the invention.

Typically, selective hybridization occurs when two nucleic acid sequences are substantially complementary (at least about 65% complementary over a stretch of at least 14 to 25 nucleotides, preferably at least about 75%, more preferably at least about 90% complementary). See Kanehisa, M., 1984, Nucleic Acids Res. 12: 203, incorporated herein by reference. As a result, it is expected that a certain degree of mismatch at the priming site is tolerated. Such mismatch may be small, such as a mono-, di- or tri-nucleotide. Alternatively, it may encompass loops, which are defined as regions in which there exists a mismatch in an uninterrupted series of four or more nucleotides.

Numerous factors influence the efficiency and selectivity of hybridization of the primer to a second nucleic acid molecule. These factors, which include primer length, nucleotide sequence and/or composition, hybridization temperature, buffer composition and potential for steric hindrance in the region to which the primer is required to hybridize, wih be considered when designing ohgonucleotide primers according to the invention.

A positive correlation exists between primer length and both the efficiency and accuracy with which a primer wih anneal to a target sequence. In particular, longer sequences have a higher melting temperature (T^_j) than do shorter ones, and are less likely to be repeated within a given target sequence, thereby minimizing promiscuous hybridization. Primer sequences with a high G-C content, or that comprise palindromic sequences tend to self-hybridize, as do their intended target sites, since unimolecular, rather than bimolecular, hybridization kinetics are generally favored in solution. t However, it is also important to design a primer that contains sufficient numbers of G-C nucleotide pairings since each G-C pair is bound by three hydrogen bonds, rather than the two that are found when A and T bases pah to bind the target sequence, and therefore forms a tighter, stronger bond. Hybridization temperature varies inversely with primer annealing efficiency, as does the concentration of organic solvents, e.g. formamide, that might be included in a priming reaction or hybridization mixture, while increases in salt concentration facilitate binding. Under stringent annealing conditions, longer hybridization probes (of use, for example, in Northern analysis), or synthesis primers, hybridize more efficiently than do shorter ones, which are sufficient under more permissive conditions. Stringent hybridization conditions typically include salt concentrations of less than about IM, more usually less than about 500 mM and preferably less than about 200 mM. Hybridization temperatures range from as low as 0°C to greater than 22°C, greater than about 30°C, and (most often) in excess of about 37°C. Longer fragments may require higher hybridization temperatures for specific hybridization. As several factors affect the stringency of hybridization, the combination of parameters is more important than the absolute measure of a single factor. Ohgonucleotide primers can be designed with these considerations in mind and synthesized according to the following methods.

1. Ohgonucleotide Primer Design Strategy The design of a particular ohgonucleotide primer for the purpose of sequencing or PCR involves selecting a sequence that is capable of recognizing the target sequence, but has a minimal predicted secondary structure. The ohgonucleotide sequence binds only to a single site in the target nucleic acid. Furthermore, the Tm of the ohgonucleotide is optimized by analysis of the length and GC content of the ohgonucleotide. Furthermore, when designing a PCR primer useful for the amphfication of genomic DNA, the selected primer sequence does not demonstrate significant matches to sequences in the GenBank database (or other available databases).

The design of a primer is facihtated by the use of readily available computer programs, developed to assist in the evaluation of the several parameters described above and the optimization of primer sequences. Examples of such programs are "Primer Select" of the DNAStar™ software package (DNAStar, Inc. ; Madison, WI), OLIGO 4.0 (National Biosciences, Inc.), PRIMER,

Ohgonucleotide Selection Program, PGEN and Amplify (described in Ausubel et al, 1995, Short Protocols in Molecular Biology, 3rd Edition, John Wiley & Sons). Primers are designed with sequences that serve as targets for other primers to produce a PCR product that has known sequences on the ends which serve as targets for further amphfication (e.g. to sequence the PCR product). If many different genes are amplified with specific primers that share a common 'tail' sequence', the PCR products from these distinct genes can subsequently be sequenced with a single set of primers. Alternatively, in order to facihtate subsequent cloning of amplified sequences, primers are designed with restriction enzyme site sequences appended to their 5' ends. Thus, all nucleotides of the primers are derived from gene sequences or sequences adjacent to a gene, except for the few nucleotides necessary to form a restriction enzyme site. Such enzymes and sites are weh known in the art. If the genomic sequence of a gene and the sequence of the open reading frame of a gene are known, design of particular primers is well within the skill of the art.

2. Synthesis

The primers themselves are synthesized using techniques which are also weh known in the art. Once designed, oligonucleotides are prepared by a suitable method, e.g. the phosphoramidite method described by Beaucage and Carruthers (1981, Tetrahedron Lett.. 22:1859) or the triester method according to Matteucci et al. (1981, J. Am. Chem. Soc, 103:3185), both incorporated herein by reference, or by other chemical methods using either a commercial automated ohgonucleotide synthesizer (which is commercially available) or VLSIPS™ technology.

B. Production of a Polynucleotide Sequence

The invention discloses polynucleotide sequences comprising polymorphisms. The polynucleotide sequences of the invention are specificaUy hybridizable to a mutant form of a gene and are therefore useful for discriminating between a wild-type form of a gene and a mutant form of a gene. The polynucleotide sequences of the invention may also be useful for expression of the encoded protein or a fragment thereof. The invention also features antisense polynucleotide sequences complementary to polynucleotide sequences comprising polymorphisms. Antisense polynucleotide sequences are useful according to the invention for inhibiting expression of an allelic form of a gene. The present invention utilizes polynucleotide sequences and fragments comprising RNA, cDNA, genomic DNA, synthetic forms, and mixed polymers. The invention includes both sense and antisense strands of the polynucleotide sequences. According to the invention, the polynucleotide sequences may be chemically or biochemically modified or may contain non-natural or derivatized nucleotide bases. Such modifications include, for example, labels, methylation, substitution of one or more of the naturally occurring nucleotides with an analog, internucleotide modifications such as uncharged linkages (e.g. methyl phosphonates,- phosphorodithioates. etc.), pendent moieties (e.g., polypeptides), intercalators, (e.g. acridine, psoralen, etc.) chelators, alkylators, and modified linkages (e.g. alpha anomeric nucleic acids, etc.) Also included are synthetic molecules that mimic polynucleotides in their abihty to bind to a designated sequence via hydrogen bonding and other chemical interactions. Such molecules are known in the art and include, for example, those in which peptide linkages substitute for phosphate linkages in the backbone of the molecule.

The polynucleotide may be a naturally occurring polynucleotide, or may be a structurally related variant of such a polynucleotide having modified bases and/or sugars and/or linkages. The term "polynucleotide" as used herein is intended to cover ah such variants.

Modifications, which may be made to the polynucleotide may include (but are not limited to) the f oho wing types: a) Backbone modifications i) phosphorothioates (X or Y or W or Z = S or any combination of two or more with the remainder as 0). e.g. Y=S (Stein et al, 1988, Nucleic Acids Res.. 15:3209), X=S (Cosstick and Vyle, 1989, Tetrahedron Letters. 30:4693), Y and Z=S (Brill et al, 1989, J. Amer. Chem. Soc. 111:2321) ii) methylphosphonates (eg Z=methyl (Miller et al, 1980, J. Biol Chem.. 255:9569)) hi) phosphoramidates (Z = N-(alkyl)₂ .g. alkyl methyl, ethyl butyl) (Z=morρholine or piperazine) (Agrawal et al, 1988, Proc. Natl Acad. Sci.. USA, 85;7079) (X or W = NH) (Mag and Engels. 1988, Nucleic Acids Res., 16:3525) iv) phosphotriesters (Z=O-alkyl e.g. methyl, ethyl etc) (Miller et al, Biochemistry, 21:5468) v) phosphorus-free linkages (e.g. carbamate, acetamidate, acetate) (Gait et al, 1974, J

ChemSo Perkin 1, 1684, Gait et al, 1979, J Chem.Soc Perkin 1, 1389) b) Sugar modifications i) 2'-deoxynucleosides (R=H) ii) 2'-O-methylated nucleosides (R=OMe) (Sproat et al, 1989, Nucleic Acids Res., 17: 3373) hi) 2'-fluoro-2'-deoxynucleosides (R=F) (Krug et al, 1989, Nucleosides and Nucleotides, 8:1473) c) Base modifications - (for a review see Jones, 1979, Int. J. Biolog. Macromolecules, 1:194) i) pyrimidine derivatives substituted in the 5-position (e.g. methyl, bromo, fluoro etc) or replacing a carbonyl group by an amino group (Piccirilh et al, 1990, Nature, 343 :33). ii) purine derivatives lacking specific nitrogen atoms (e.g. 7-deaza adenine, hypoxantliine) or functionalized in the 8-position (e.g. 8-azido adenine, 8-bromo adenine) d) Polynucleotides covalently hnked to reactive flunctional groups, e.g. : i) psoralens (Miher et al, 1988, Nucleic Acids Res. Special Pub. No. 20:113, phenanthrolines (Sun et al,- 1988, Biochemistry. 27:6039), mustards (Vlassov et al, 1988, Gene, 72:313) (irreversible cross-linking agents with or without the need for co-reagents) ii) acridine (intercalating agents) (Helene et al, 1985, Biochimie, 67:777) hi) thiol derivatives (reversible disulphide formation with proteins) (Connolly and Newman, 1989. Nucleic Acids Res., 17:4957) iv) aldehydes (Schiffs base formation) v) azido, bromo groups (UV cross-linking) vi) elhpticines (photolytϊc cross-linking) (Perrouault et al, 1990, Nature, 344:358) e) Polynucleotides covalently linked to hpophihc groups or other reagents capable of improving uptake by cells, e.g.: i) cholesterol (Letsinger et al, 1989, Proc. Natl. Acad. Sci. USA, 86:6553), polyamines

(Lemaitre et al., 1987, Proc Natl Acad. Sci. USA, 84: 648), other soluble polymers (e.g. polyethylene glycol) f) Polynucleotides containing alpha-nucleosides (Morvan et al., Nucleic Acids Res., 15: 3421) g) Combinations of modifications a)-f)

It should be noted that such modified polynucleotides, while sharing features with polynucleotides designed as "anti-sense" inhibitors, are distinct in that the compounds correspond to sense-strand sequences and the mechanism of action depends on protein-nucleic acid interactions and does not depend upon interactions with nucleic acid sequences.

1. Polynucleotide Sequences Comprising DNA a. Cloning Polynucleotide sequences comprising DNA can be isolated from cDNA or genomic hbraries (including YAC and BAC hbraries) by cloning methods weh known to those skihed in the art (Ausubel et al, supra). Briefly, isolation of a DNA clone comprising a particular polynucleotide sequence involves screening a recombinant DNA or cDNA hbrary and identifying the clone containing the deshed sequence. Cloning wih involve the fohowing steps. The clones of a particular hbrary are spread onto plates, transferred to an appropriate substrate for screening, denatured, and probed for the presence of a particular sequence. A description of hybridization conditions, and methods for producing labeled probes is included below.

The deshed clone is preferably identified by hybridization to a nucleic acid probe or by expression of a protein that can be detected by an antibody. Alternatively, the deshed clone is identified by polymerase chain amphfication of a sequence defined by a particular set of primers according to the methods described below.

The selection of an appropriate hbrary involves identifying tissues or cell lines that are an abundant source of the deshed sequence. Furthermore, if the polynucleotide sequence of interest contains regulatory sequence or intronic sequence a genomic hbrary is screened (Ausubel et al, supra). b. Genomic DNA

Polynucleotide sequences of the invention are amplified from genomic DNA. Genomic DNA is isolated from tissues or cells according to the fohowing method.

To facihtate detection of a variant form of a gene from a particular tissue, the tissue is isolated free from surrounding normal tissues. To isolate genomic DNA from mammalian tissue, the tissue is minced and frozen in hquid nitrogen. Frozen tissue is ground into a fine powder with a prechihed mortar and pestle, and suspended in digestion buffer (100 mM NaCl, 10 mM TrisCl, pH 8.0, 25 mM EDTA, pH 8.0, 0.5% (w/v) SDS, 0.1 mg/ml proteinase K) at 1.2ml digestion buffer per lOOmg of tissue. To isolate genomic DNA from mammalian tissue culture cells, cells are pelleted by centrifugation for 5 min at 500 x g, resuspended in 1-10 ml ice-cold PBS, repeheted for 5 min at 500 x g and resuspended in 1 volume of digestion buffer.

Samples in digestion buffer are incubated (with shaking) for 12-18 hours at 50°C, and then extracted with an equal volume of phenol/chloroform/isoamyl alcohol. If the phases are not resolved fohowing a centrifugation step (10 min at 1700 x g), another volume of digestion buffer (without proteinase K) is added and the centrifugation step is repeated. If a thick white material is evident at the interface of the two phases, the organic extraction step is repeated. Fohowing extraction the upper, aqueous layer is transferred to a new tube to which will be added 1/2 volume of 7.5M ammomum acetate and 2 volumes of 100% ethanol. The nucleic acid is pelleted by centrifugation for 2 min at 1700 x g, washed with 70% ethanol, ah dried and resuspended in TE buffer (10 mM TrisCl, pH 8.0, 1 mM EDTA, pH 8.0) at lmg/ml. Residual RNA is removed by incubating the sample for 1 hour at 37°C in the presence of 0.1 % SDS and 1 mg/ml DNAse-free RNASE, and repeating the extraction and ethanol precipitation steps. The yield of genomic DNA, according to this method is expected to be approximately 2 mg DNA/1 g cells or tissue (Ausubel et al, supra). Genomic DNA isolated according to this method can be used for Southern blot analysis, restriction enzyme digestion, dot blot analysis or PCR analysis, according to the invention. c Restriction digest (of cDNA or genomic DNA) Fohowing the identification of a deshed cDNA or genomic clone containing a particular sequence, polynucleotides of the invention are isolated from these clones by digestion with restriction ' enzymes.

The technique of restriction enzyme digestion is weh known to those skihed in the art (Ausubel et al, supra). Reagents useful for restriction enzyme digestion are readily available from commercial vendors including New England Biolabs, Boebringer Mannheim, Promega, as weh as other sources. d. PCR Polynucleotide sequences of the invention are amplified from genomic DNA or other natural sources by the polymerase chain reaction (PCR). PCR methods are well-known to those skihed in the art.

PCR provides a method for rapidly amphfying a particular DNA sequence by using multiple cycles of DNA rephcation catalyzed by a thermostable, DNA-dependent DNA polymerase to amplify the target sequence of interest. PCR requires the presence of a nucleic acid to be amplified, two single stranded ohgonucleotide primers flanking the sequence to be amplified, a DNA polymerase, deoxyribonucleoside triphosphates, a buffer and salts.

The method of PCR is weh known in the art. PCR, is performed as described in Mulhs and Faloona, 1987, Methods Enzymol, 155: 335, herein incorporated by reference.

PCR is performed using template DNA (at least 1 fg; more usefully, 1 - 1000 ng) and at least 25 pmol of ohgonucleotide primers. A typical reaction mixture includes: 2 ml of DNA, 25 pmol of ohgonucleotide primer, 2.5 ml of lOx PCR buffer 1 (Perkin-Elmer, Foster City, CA), 0.4 ml of 1.25 mM dNTP, 0.15 ml (or 2.5 units) of Taq DNA polymerase (Perkin Elmer, Foster City, CA) and deionized water to a total volume of 25 ml. Mineral oil is overlaid and the PCR is performed using a programmable thermal cycler.

The length and temperature of each step of a PCR cycle, as weh as the number of cycles, are adjusted according to the stringency requirements in effect. Annealing temperature and timing are determined both by the efficiency with which a primer is expected to anneal to a template and the degree of mismatch that is to be tolerated. The abihty to optimize the stringency of primer annealing conditions is weh within the knowledge of one of moderate skill in the art. An annealing temperature of between 30°C and 72°C is used. Initial denaturation of the template molecules normally occurs at between 92°C and 99°C for 4 minutes, fohowed by 20-40 cycles consisting of denaturation (94-99°C for 15 seconds to 1 minute), annealing (temperature determined as discussed above; 1-2 minutes), and extension (72°C for 1 minute). The final extension step is generahy carried out for 4 minutes at 72°C, and may be fohowed by an indefinite (0-24 hour) step at 4°C.

Several techniques for detecting PCR products quantitatively without electrophoresis may be useful according to the invention in order to make it more suitable for easy clinical use. One of these techniques, for which there are commercially available kits such as Taqman™ (Perkin Elmer, Foster City, CA), is performed with a transcript-specific antisense probe. This probe is specific for the PCR product (e.g. a nucleic acid fragment derived from a gene) and is prepared with a quencher and fluorescent reporter probe complexed to the 5' end of the ohgonucleotide. Different fluorescent markers can be attached to different reporters, allowing for measurement of two products in one reaction. When Taq DNA polymerase is activated, it cleaves off the fluorescent reporters of the probe bound to the template by virtue of its 5'-to-3 ' nucleolytic activity. In the absence of the quenchers, the reporters now fluoresce. The color change in the reporters is proportional to the amount of each specific product and is measured by a fluorometer; therefore, the amount of each color can be measured and the PCR product can be quantified. The PCR reactions can be performed in 96 weh plates so that samples derived from many individuals can be processed and measured simultaneously. The Taqman™ system has the additional advantage of not requiring gel electrophoresis and ahows for quantification when used with a standard curve. 2. Polynucleotide Sequences Comprising RNA The present invention also provides a polynucleotide sequence comprising RNA. A polynucleotide comprising RNA is useful for detecting snps and polymorphisms by tecliniques including but not limited to hybridization methods or the RNase protection method. A polynucleotide comprising RNA is also useful as a template for the in vitro production of protein. A polynucleotide comprising RJSf A is also useful for detecting and locahzing specific mRNA sequences by in situ hybridization.

Polynucleotide sequences comprising RNA can be produced according to the method of in vitro transcription.

The technique of in vitro transcription is weh known to those of skill in the art. Briefly, the gene of interest is inserted into a vector containing an SP6, T3 or T7 promoter. The vector is linearized with an appropriate restriction enzyme that digests the vector at a single site located downstream of the coding sequence. Fohowing a phenol/chloroform extraction, the DNA is ethanol precipitated, washed in 70% ethanol, dried and resuspended in sterile water. The in vitro transcription reaction is performed by incubating the linearized DNA with transcription buffer (200 mM TrisCl, pH 8.0,40 mM MgCl₂, 10 mM spermidine, 250 NaCl [T7 or T3] or 200 mM TrisCl, pH 7.5,30 mM MgC^, lOmM sper idine [SP6]), ditMothreitol, RNASE inhibitors, each of the four ribonucleoside triphosphates, and either SP6, T7 or T3 RNA polymerase for 30 min at 37°C. To prepare a radiolabeled polynucleotide comprising RNA, unlabeled UTP wih be omitted and -SUTP wih be included in the reaction mixture. ,^•• The DNA template is then removed by incubation with DNasel. Fohowing ethanol precipitation, an aliquot of the radiolabeled RNA is counted in a scintihation counter to determine the cpm/ml (Ausubel et al, supra). V

Alternatively, polynucleotide sequences comprising RNA are prepared by chemical synthesis techniques such as solid phase phosphoramidite (described above).

3. Polynucleotide Sequences Comprising Ohgonucleotides A polynucleotide sequence comprising ohgonucleotides can be made by using ohgonucleotide synthesizing machines which are commercially available (described above).

4. Polynucleotide Sequences Encoding Fusion Proteins Polynucleotide sequences of the invention can be used to express the protein product (or fragment thereof) of the gene of interest by inserting the polynucleotide sequence into an expression vector. Expression vectors suitable for protein expression in mammalian cehs, bacterial cehs, insect cehs or plant cehs are weh known in the art and are described in Section H entitled "Production of a Mutant Protein".

Polynucleotide sequences of the invention can be used to prepare hybrid polynucleotides comprising a sequence of a gene adjacent to a sequence encoding a foreign protein or a fragment thereof (e.g lacZ, trpE, glutathionine S-transferase or thioredoxin) or a protein tag (hemmaglutinin or FLAG). Such hybrid polynucleotides produce fusion proteins that are useful, according to the invention, for improved expression and/or rapid isolation of a protein or protein fragment, encoded by the sequence of a gene. Hybrid polynucleotides are also useful as a source of antigen for the production of antibodies.

Nucleic acid constructs comprising a polynucleotide of genomic, cDNA, synthetic or semi- synthetic origin in association with a polynucleotide sequence encoding a foreign protein or a fragment thereof, (carrier sequence) can be generated by recombinant nucleic acid techniques weh known in the art (See Ausubel et al, supra). According to this method, the cloned gene is introduced into an expression vector at a position located 3' to a carrier sequence coding for the amino terminus of a highly expressed protein, an entire functional moiety of a highly expressed protein or the entire protein. It is preferable to use a earner sequence from an E. coli gene or from any gene that is expressed at high levels in E. coli. It is often preferable to select a carrier sequence that wih facihtate protein purification, either with antibodies, or with an affinity purification protocol that is specific for the carrier protein being used. For example, the purification protocol can be designed in accordance with the unique physical properties of the carrier protein (e.g. heat stabihty). Alternatively, the tag sequence, may encode a protein (e.g. glutathione-S -transferase (GST)) which can be purified by either a chemical interaction (for example glutathione purification of GST). Alternatively, some carrier proteins, such as thioredoxin (Trx) can be selectively released from intact cehs by osmotic shock or freeze/thaw procedures. Often, proteins that are fused to these carrier proteins can be purified away from intracellular contaminants by virtue of the physical attributes of the carrier protein (Ausubel et al, supra).

To ensure that a fusion protein is useful, according to the invention, it may be necessary to modify the expression protocol to produce a soluble protein. Due to the fact that high-level expression of certain proteins can lead to the formation of inclusion bodies, if a soluble protein is required it may be necessary to modify the fohowing variables. The temperature at which expression is induced can affect inclusion body formation since inclusion body formation is induced at higher temperatures (37°C and 42°C) and inhibited at lower temperatures (30°C). In certain instances, lowering the total level of protein expression can lead to an increase in the proportion of soluble protein that is produced. The strain background of the cehs in which the protein is being produced can affect the proportion of a particular protein that is expressed in a soluble form. Furthermore, the choice of carrier protein can affect the solubility of an expressed fusion protein (Ausubel et al, supra). An additional problem that can be encountered when producing fusion proteins in E. coli is formation of an unstable protein, or a protein that is cleaved at the site of the junction between the carrier sequence and the sequence of the protein of interest. To decrease complications due to protein instabihty one can arrange for the fusion protein to be expressed as insoluble aggregates. Alternatively, one can express the fusion protein in E. coli strains that are deficient in proteases (Ausubel et al, supra).

Often it is useful to remove the carrier protein moiety from the protein of interest to facihtate biochemical and functional analyses. Methods for cleavage of fusion proteins to remove the carrier are known to those skihed in the art. The choice of a method is usuahy determined by the composition, sequence, and physical characteristics of the particular protein. Reagents such as cyanogen bromide, hydroxylamine or low pH can be used to chemically cleave fusion proteins. To avoid complications resulting from chemical cleavage (e.g. the presence of chemical cleavage sites in the protein of interest and/or the occunence of side reactions resulting in protein modification), enzymatic cleavage methods can be used. Enzymatic cleavage protocols are advantageous because they can be carried out under relatively mild reaction conditions, and because they involve highly specific cleavage reactions. Enzymes useful for enzymatic cleavage of fusion proteins include factor Xa, thrombin, enterokinase, renin and collagenase (Ausubel et al, supra).

Recombinant constructs encoding fusion proteins wherein the carrier sequence is on the order of 9-15 codons, can be generated by PCR methods. According to this method, a PCR primer wih be designed to contain at least 13 nucleotides that are identical to the target sequence on either side of the nucleotide sequence encoding the carrier sequence. Preferably, the PCR primer wih also contain a restriction enzyme site to facihtate cloning of the amplified product into an appropriate expression vector. PCR wih be carried out as described above and the sequence of the amplified product wih be confirmed by sequence analysis as described in Section D entitled "Isolation of a Wild type Gene". Alternatively, recombinant constructs encoding fusion proteins can be generated by site/ohgonucleotide directed mutatagenesis (Ausubel et al., supra). According to the method of site directed mutatagenesis the DNA to be mutated is inserted into a plasmid which has an FI origin of replication. A mutagenesis ohgonucleotide is designed to contain 13 bp that are 100% identical to the target sequence, on either side of a sequence coding for the 9-15 codons of carrier sequence that is to be added by the mutatgenesis protocol.

A single stranded preparation of the vector is prepared by the fohowing method. Fohowing transformation of an appropriate bacterial strain (e.g. CJ236) with the recombinant plasmid and plating of the bacteria on LB agar plates, a single resulting colony is grown in 4x5 ml of LB plus ampicihin for 1 hour at 37°C with vigorous shaking. M13K07 helper phage (2 ml, approximately lO^-lO¹¹ plaque forming units) is added and the bacteria are grown for an additional hour at 37°C with vigorous shaking. Following the addition of 7 ml of kanamycin (50 mg/ml), the bacteria are grown overnight at 37°C with vigorous shaking. The fohowing day bacterial cultures are pooled and cehs are separated by centrifugation. After the addition of 2.6 ml of 20% polyethylene glycol 200-800/2M NaCl to 20 ml of bacterial supernatant, the sample is incubated for 1 - 1.5 hours on ice. The sample is pelleted by centrifugation at 9000 rpm for 20 minutes. Fohowing removal of the supernatant, residual supernatant are removed by centrifugation at 3000 rpm for 5 minutes. The pellet is resuspended in 400 ml of TE, extracted twice with phenol and four times with phenolchloroform and ethanol precipitated. The resulting pellet is resuspended in 40 ml TE.

Mutagenesis is performed by using a muta-genekit (Bio-Rad, Hercules, CA) according to the fohowing method. To kinase the ohgonucleotide primer, 1 ml (200ng) of ohgonucleotide is incubated in the presence of 2 ml of 10 kinase buffer (0.5M Tris, pH 8.0, 70mM MgCl^, lOmM DTT), 2 ml lOmM rATP, 2 ml polynucleotide kinase and 13 ml I 0 for 37°C for 1 hour. To carry out the annealing and synthesis steps, 2.5 ml of single-stranded template are mixed with 1 ml of kinased ohgonucleotide, 1.0 ml of 10X annealing buffer (200mM Tris-HCl, pH 7.4, 20 mM MgCl₂, 500mM NaCl) and 5.5 ml FLO for 10 min at 65°C. The reaction mixture is slow-cooled to 37°C. Once the sample has reached 37°C, the sample is spun briefly in a microfuge. Fohowing the addition of 1.0 ml of 10X synthesis buffer (5mM each dATP, dCTP, cGTP, dTTP, lOmM ATP, lOOmM Tris-HCl, pH 7.4, 50 mM MgCl., 20mM DTT), 1.0 ml T4 DNA hgase and 0.5 ml of T4 DNA polymerase, the sample is incubated for 5 minutes on ice, 5 minutes at room temperature and 1 hour at 37°C. A 2 ml ahquot of the sample is used to transform E. coli.

DNA is isolated from the transformed E. coli cehs by mini prep methods known in the art (Ausubel et al, supra), and sequenced according to methods known in the art (described in Section D entitled "Isolation of a Wild Type Gene".

C. Production of a Nucleic acid Probe

The invention discloses nucleic acid probes. Preferably, the nucleic acid probes of the invention are specifically hybridizable to a mutant gene but not to a wild type form of a gene due to the presence of one or more polymorphisms. These ahele specific probes can be used to screen DNA sequences of a gene which have been amplified by PCR, or are present in a genomic DNA or RNA test sample. Hybridization of a particular ahele specific probe to an amplified gene sequence, under stringent conditions (described below), indicates that the polymorphism contained in the probe is present in the amplified sequence. Hybridization of a particular ahele specific probe to a test sample comprising genomic DNA or RNA, under stringent conditions (described below), indicates that the polymorphism contained in the probe, is present in the nucleic acid of the test sample. Nucleic acid probes that are specifically hybridizable to a wild type form of a gene but not to a mutant form of a gene are also useful according to the invention.

In another embodiment, the probes of the claimed invention will be specific for a nucleic acid region that is adjacent to a region that is thought to contain one or more polymorphisms. These probes wih be useful for detecting the presence of one or more polymorphisms in the adjacent region by the method of primer extension (as described in Section F entitled "Identification and Characterization of Polymorphisms".

In other embodiments, probes of the claimed invention wih be used to detect a gain or loss of a restriction enzyme site known to contain one or more polymorphisms of the claimed invention. Nucleic acid probes, according to this embodiment, are able to detect a restriction enzyme fragment that is of a size that can be easily separated on an agarose gel and visualized by Southern blot analysis. Probes that are useful according to this embodiment of the claimed invention can be specific for any region within a gene or outside of a gene.

The nucleic acids probes of the invention are useful for a variety of hybridization-based analyses including but not limited to Southern hybridization to genomic DNA, cDNA sequences or PCR amphfication^'products, Northern hybridization to mRNA and RNase protection assays, DNA ^; sequencing and isolation of genomic or cDNA clones of a gene. The probes may also be used to determine whether mRNA encoded for by a gene is present in a ceh or tissue by the method of in situ hybridization. These techniques are weh known in the art and can be performed as described in Ausubel et al, supra.

According to the methods of the above-referenced hybridization assays, polymorphisms associated with aheles of a gene, which either predispose to a particular disease (e.g. osteoarthritis) or are not associated with a particular disease (e.g. osteoarthritis), wih be detected by the formation of a stable hybrid consisting of a polynucleotide probe comprising one or more polymorphisms and a target sequence, that also comprises one or more polymorphisms, under stringent to moderately stringent hybridization and wash conditions. If it is expected that the probes wih be perfectly complementary to the target sequence, stringent conditions wih be used. Hybridization stringency may be lessened if some mismatching is expected, for example, if variants are expected with the result that the probe wih not be completely complementary. Conditions are chosen which rule out nonspecific/adventitious bindings, that is, which minimize noise. Since such indications identify neutral DNA polymorphisms as weh as mutations, these indications need further analysis (such as assays described in Section F entitled "Identification and Characterization of Polymorphisms") to demonstrate detection of a susceptibihty ahele of a gene.

Probes for aheles of a gene may be derived from genomic DNA or cDNA sequences from specific for the gene of interest. The probes may be of any suitable length, which span ah or a portion of the region containing the gene. If the target sequence contains a sequence identical to that of the probe, the probes may be short, e.g., in the range of about 8-30 base pahs, since the hybrid wih be relatively stable under even stringent conditions. If some degree of mismatch is expected with the probe, i.e., if it is suspected that the probe wih hybridize to a variant region, a longer probe maybe employed which hybridizes to the target sequence with the requisite specificity.

Probes according to the invention also include an isolated polynucleotide attached to a label or a reporter molecule which may be useful for isolating other polynucleotide sequences, having sequence similarity by standard methods, including but not limited to the above-referenced hybridization-based assays. Techniques for preparing and labeling probes (as described in Ausubel et al. Supra) are included below. A wide variety of labels and conjugation techniques are known by those skihed in the art and can be used in a various nucleic acid and amino acid assays. Means for producing labeled hybridization or PCR probes for detecting related sequences include ohgolabeling, nick translation, end-labeling or PCR amphfication using a labeled nucleotide. Alternatively, the protein- encoding sequence, or any portion of it, may be cloned into a vector for the production of an mRNA probe. Such vectors are known in the art, are commercially available, and may be used to synthesize RNA probes in vitro by addition of an appropriate RNA polymerase such as T7, T3 or SP6 and labeled nucleotides.

A number of companies such as Pharmacia Biotech (Piscataway NJ), Promega (Madison WI) and US Biochemical Corp (Cleveland OH) supply commercial kits and protocols for these procedures. Suitable reporter molecules or labels include those radionuchdes, enzymes, fluorescent, chemiluminescent, or chromogenic agents as weh as substrates, cofactors, inhibitors, magnetic particles and the like. Patents teaching the use of such labels include US Patents 3,817,838; 3,350,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149 and 4,366,241. Also, recombmant immunoglobulins maybe produced as shown in US Patent No. 4,816,567 incorporated herein by reference. Probes comprising synthetic ohgonucleotides or other polynucleotides of the present invention may be derived from naturally occurring or recombinant single- or double- stranded polynucleotides, or be chemically synthesized.

Portions of the polynucleotide sequence having at least approximately 5 nucleotides, preferably 9-15 nucleotides, fewer than about 6 kb and usually fewer than about 1 kb, from a polynucleotide sequence encoding a gene are preferred as probes.

A DNA probe useful according to the present invention can be isolated from a gene or a polynucleotide constmct derived from a gene, or from a cDNA sequence specific for a gene or a cDNA construct specific for a gene by the methods of PCR or restriction enzyme digestion, as described above. Riboprobes useful according to the invention can be synthesized by the method of in vitro transcription, or by chemical synthesis methods, as described above.

An ohgonucleotide probe useful according to the invention can be designed, as described above, and synthesized in a commerciahy available automated synthesizer. Nucleic acid hybridization rate and stability wih be affected by a variety of experimental parameters including salt concentration, temperature, the presence of organic solvents, the viscosity of the hybridization solution, the base composition of the probe, the length of the duplex, and the number of mismatches between the hybridizing nucleic acids (Ausubel et al, supra), and as described in Section A entitled "Design and Synthesis of Ohgonucleotide Primers". Southern blot analysis can be used to detect sequence variations in a gene from a PCR amplified product or from a total genomic DNA test sample via a non-PCR based assay. The method of Southern blot analysis is weh known in the art (Ausubel et al, supra, Sambrook et al, 1989, Molecular Cloning. A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY). This, technique involves the transfer of DNA fragments from an electrophoresis gel to a membrane support resulting in the immobilization of the DNA fragments. The resulting membrane carries a semipermanent reproduction of the banding pattern of the gel.

Southern blot analysis is performed according to the fohowing method. Genomic DNA (5-20 mg) is digested with the appropriate restriction enzyme and separated on a 0.6-1.0% agarose gel in TAE buffer. The DNA is transfened to a commerciahy available nylon or nitrocehulose membrane (e.g. Hybond-N membrane, Amersham, Arlington Heights, IL) by methods weh known in the art

(Ausubel et al, supra, Sambrook et al, supra). Fohowing transfer and UV cross linking, the membrane is hybridized with a radiolabeled probe in hybridization solution (e.g. under stringent conditions in 5X SSC, 5XDenhardt solution, 1% SDS) at 65°C. Alternatively, high stringency hybridization can be performed at 68°C or in a hybridization buffer containing a decreased concentration of salt, for example 0. IX SSC. The hybridization conditions can be varied as necessary according to the parameters described in Section A entitled "Design and Synthesis of Ohgonucleotide Primers". Fohowing hybridization, the membrane is washed at room temperature in 2X SSC/0.1% SDS and at 65°C in 0.2X SSC/0.1% SDS, and exposed to film. The stringency of the wash buffers can also be varied depending on the amount of the background signal (Ausubel et al, supra).

Detection of a nucleic acid probe-target nucleic acid hybrid will include the step of hybridizing a nucleic acid probe to the DNA target. This probe may be radioactively labeled or covalently hnked to an enzyme such that the covalent linkage does not interfere with the specificity of the hybridization. A resulting hybrid can be detected with a labeled probe. Methods for radioactively labeling a probe include random ohgonucleotide primed syntliesis, nick translation or kinase reactions (see Ausubel et al, supra). Alternatively, a hybrid can be detected via non-isotopic methods. Non-isotopicahy labeled probes can be produced by the addition of biotin or digoxigenin, fluorescent groups, chenhluminescent groups (e.g. dioxetanes, particularly triggered dioxetanes), enzymes or antibodies. Typically, non- isotopic probes are detected by fluorescence or enzymatic methods. Detection of a radiolabeled probe-target nucleic acid complex can be accomplished by separating the complex from free probe and measuring the level of complex by autoradiography or scintihation counting. If the probe is covalently linked to an enzyme, the enzyme-probe-conjugate- target nucleic acid complex wih be isolated away from the free probe enzyme conjugate and a substrate wih be added for enzyme detection. Enzymatic activity wih be observed as a change in color development or luminescent output resulting in a 10³-10⁵ increase in sensitivity. An example of the preparation and use of nucleic acid probe-enzyme conjugates- as hybridization probes (wherein the enzyme is alkaline phosphatase) is \ described in (Jablonski et al, 1986, Nucleic Acids Res., 14:6115)

Two-step label amphfication methodologies are known in the art. These assays are based on the principle that a smah hgand (such as digoxigenin, biotin, or the like) is attached to a nucleic acid probe capable of specifically binding to a gene. Ahele specific gene probes are also useful according to this method.

According to the method of two-step label amphfication, the smah hgand attached to the nucleic acid probe wih be specifically recognized by an antibody-enzyme conjugate. For example, digoxigenin wih be attached to the nucleic acid probe and hybridization wih be detected by an antibody- alkaline phosphatase conjugate wherein the alkaline phosphatase reacts with a chenhluminescent substrate. For methods of preparing nucleic acid probe-smah hgand conjugates, see (Martin et al, 1990, BioTechniques, 9:762). Alternatively, the smah hgand wih be recognized by a second hgand- enzyme conjugate that is capable of specifically complexing to the first hgand. A weh known example of this manner of smah hgand interaction is the biotin avidin interaction. Methods for labeling nucleic acid probes and their use in biotin-avidin based assays are described in Rigby et al, 1977, J. Mol Biol, 113:237 and Nguyen et al, 1992, BioTechniques. 13:116).

Variations of the basic hybrid detection protocol are known in the art, and include modifications that facihtate separation of the hybrids to be detected from extraneous materials and/or that employ the signal from the labeled moiety. A number of these modifications are reviewed in, e.g., Matthews & Kricka, 1988, Anal Biochem.. 169:1; Landegren et al, 1988, Science, 242:229; Mittlin, 1989, Clincal Chem. 35:1819; U.S. Pat. No. 4,868,105, and in EPO Publication No. 225,807.

D. Isolation of a Wild type gene

A wild type version of a candidate gene according to the invention can be isolated by cloning from an appropriately selected genomic hbrary according to methods weh known in the art. Methods of cloning are described in Section B entitled "Production of a Polynucleotide Sequence The sequence of the cloned gene wih be determined by sequencing methods weh known in the art (see Ausubel et al, supra and Sambrook et al, supra). Methods of sequencing employ such enzymes as the Klenow fragment of DNA polymerase I, Sequenase® (US Biochemical Corp, Cleveland, OH), Taq polymerase (Perkin Elmer, Norwalk, CT), thermostable T7 polymerase (Amersham, Chicago, IL), or combinations of recombinant polymerases and proofreading exonucleases such as the ELONGASE Amphfication System (Gibco BRL, Gaithersburg, MD).

Preferably, the process is automated with machines such as the Hamilton Micro Lab 2200 (Hamilton, Reno NV), Peltier Thermal Cycler (PTC200; MJ Research, Watertown, MA) and the ABI 377 DNA sequencers (Perkin Elmer).

E. Isolation of a Mutant Gene

A mutant version of a candidate gene according to the invention can be isolated by cloning from an appropriately selected genomic hbrary according to methods weh known in the art. Methods of cloning are described in Section B entitled "Production of a Polynucleotide Sequence."

The sequence of the cloned gene wih be determined by sequencing methods described in Section D entitled "Isolation of a Wild Type Gene."

F. Identification and Characterization of Polymorphisms a. Identification of SNPs by in silico methods (isSNPs)

1. Identification of Polymorphisms in Candidate Genes The starting point is a set of experimentally derived nucleic acid sequences. In order to be useful for SNP discovery by the invention, it is prefeπed that the sequences have complete chromatogram files from a gel or capillary electrophoresis sequencing machine. When this is not available, quahty score data which assigns a score to each base in the sequence indicating the likelihood of eιτor for the basecah may be used. If neither of these data are available, the sequence may be used to assist the clustering of other sequences and in some cases to provide additional verification for a discovered SNP, but is not be used by the invention for the identification of the polymorphism. The population of sequences used may constitute either a database of cDNA-derived sequences or genomic sequence. In a prefeπed embodiment, sequences used by the invention are from an assembled cDNA database, such as the LifeSeqGold database (Incyte Genomics, Inc(Incyte), Palo Alto, CA).

Derivation of Nucleic Acid Sequences cDNA was isolated from hbraries constructed using RNA derived from normal and diseased human tissues and cell lines. The human tissues and cell lines used for cDNA hbrary construction were selected from a broad range of sources to provide a diverse population of cDNAs representative of gene transcription throughout the human body. Descriptions of the human tissues and ceh lines used for cDNA hbrary construction are provided in the LIFESEQ database (Incyte Pharmaceuticals, Inc. (Incyte), Palo Alto CA). Human tissues were broadly selected from, for example, cardiovascular, dermatologic, endocrine, gastrointestinal, hematopoietic/immune system, musculoskeletal, neural, reproductive, and urologic sources.

Ceh lines used for cDNA hbrary construction were derived from, for example, leukemic cehs, ^" teratocarcinomas, neuroepithehomas, cervical carcinoma, lung fibroblasts, and endothehal cehs. Such ceh lines include, for example, THP-1, Jurkat, HUVEC, hNT2, WI38, HeLa, and other ceh lines commonly used and available from pubhc depositories (American Type Culture Cohection, Manassas VA). Prior to mRNA isolation, ceh lines were untreated, treated with a pharmaceutical agent such as 5'-aza-2 -deoxycytidine, treated with an activating agent such as hpopolysaccharide in the case of leukocytic ceh lines, or, in the case of endothehal ceh lines, subjected to shear stress.

Sequencing of the cDNAs

Methods for DNA sequencing are weh known in the art. Conventional enzymatic methods employ the Klenow fragment of DNA polymerase I, SEQUENASE DNA polymerase (U.S. Biochemical Corporation, Cleveland OH), Taq polymerase (The Perkin-Elmer Corporation (Perkin- Elmer), Norwalk CT), thermostable T7 polymerase (Amersham Pharmacia Biotech, Inc. (Amersham Pharmacia Biotech), Piscataway NJ), or combinations of polymerases and proofreading exonucleases such as those found in the ELONGASE amphfication system (Life Technologies hie (Life Technologies), Gaithersburg MD), to extend the nucleic acid sequence from an ohgonucleotide primer annealed to the DNA template of interest. Methods have been developed for the use of both single- stranded and double-stranded templates. Chain termination reaction products maybe electrophoresed on urea-polyacrylamide gels and detected either by autoradiography (for radioisotope-labeled nucleotides) or by fluorescence (for fluorophore-labeled nucleotides). Automated methods for mechanized reaction preparation, sequencing, and analysis using fluorescence detection methods have been developed. Machines used to prepare cDNAs for sequencing can include the MICROLAB 2200 hquid transfer system (Hamilton Company (Hamilton), Reno NV), Peltier thermal cycler (PTC200; MJ Research, Inc. (MJ Research), Watertown MA), and ABI CATALYST 800 thermal cycler (Perkin-Elmer). Sequencing can be carried out using, for example, the ABI 373 or 377

(Perkin-Elmer) or MEGABACE 1000 (Molecular Dynamics, Inc. (Molecular Dynamics), Sunnyvale CA) DNA sequencing systems, or other automated and manual sequencing systems weh known in the art.

The nucleotide sequences have been prepared by current, state-of-the-art, automated methods and, as such, may contain occasional sequencing errors or unidentified nucleotides. Such unidentified nucleotides are designated by an N. These infrequent unidentified bases do not represent a hindrance to practicing the invention for those skilled in the art. Several methods employing standard recombinant techniques may be used to coπect errors and complete the missing sequence information. (See, e.g., those described in Ausubel, F.M. et al (1997) Short Protocols in Molecular Biology, John Wiley & Sons, New York NY; and Sambrook, J. et al. (1989) Molecular Cloning, A Laboratory Manual Cold Spring Harbor Press, Plainview NY.)

Assembly of cDNA Sequences

Human polynucleotide sequences maybe assembled using programs or algorithms weh known in the art. Sequences to be assembled are related, whohy or in part, and may be derived from a single or many different transcripts. Assembly of the sequences can be performed using such programs as PHRAP (Phils Revised Assembly Program) and the GELVLEW fragment assembly system (GCG), or other methods known in the art.

Alternatively, cDNA sequences are used as "component" sequences that are assembled into "template" or "consensus" sequences as fohows. Sequence chromatograms are processed, verified, and quality scores are obtained using PHRED. Raw sequences are edited using an editing pathway known as Block 1 (See, e.g., the LIFESEQ Assembled User Guide, Incyte Pharmaceuticals, Palo Alto, CA). A series of BLAST comparisons is performed and low-infoπnation segments and repetitive elements (e.g., dinucleoti.de repeats, Alu repeats, etc.) are replaced by "n's", or masked, to prevent spurious matches. Mitochondrial and nbosomal RNA sequences are also removed. The processed sequences are then loaded into a relational database management system (RDMS) which assigns edited sequences to existing templates, if available. When additional sequences are added into the RDMS, a process is initiated which modifies existing templates or creates new templates from works in progress (i.e., nonfinal assembled sequences) containing queued sequences or the sequences themselves. After the new sequences have been assigned to templates, the templates can be merged into bins. If multiple templates exist in one bin, the bin can be spht and the templates reannotated.

A resultant template sequence may contain either a partial or a full length open reading frame, or ah or part of a genetic regulatory element. This variation is due in part to the fact that the full length cDNAs of many genes are several hundred, and sometimes several thousand, bases in length. With current technology, cDNAs comprising the coding regions of large genes cannot be cloned because of vector limitations, incomplete reverse transcription of the mRNA, or incomplete "second strand" synthesis. Template sequences maybe extended to include additional contiguous sequences derived from the parent RNA transcript using a variety of methods known to those of skih in the art. Extension may thus be used to achieve the full length coding sequence of a gene.

Analysis of the cDNA Sequences

The cDNA sequences are analyzed using a variety of programs and algorithms which are weh known in the art. (See, e.g., Ausubel, supra. Chapter 7.7; Meyers, R.A. (Ed.) (1995) Molecular Biology and Biotechnology, Wiley VCH, New York NY, pp. 856-853). These analyses comprise both reading frame determinations, e.g., based on triplet codon periodicity for particular organisms (Fickett, J.W. (1982) Nucleic Acids Res. 10:5303-5318); analyses of potential start and stop codons; and homology searches. Computer programs known to those of skih in the art for performing computer-assisted searches for amino acid and nucleic acid sequence similarity, include, for example, Basic Local Ahgnment Search Tool (BLAST; Altschul, S.F. (1993) J. Mol. Evol. 36:290-300; Altschul, S.F.et al. (1990) J. Mol. Biol. 215:403-410.) BLAST is especially useful in determining exact matches and comparing two sequence fragments of arbitrary but equal lengths, whose ahgnment is locahy maximal and for which the ahgnment score meets or exceeds a threshold or cutoff score set by the user

(Karlin, S. et al. (1988) Proc. Natl Acad. Sci. USA 85:841-845.) Using an appropriate search tool (e.g., BLAST or HMM), GenBank, SwissProt, BLOCKS, PFAM and other databases maybe searched for sequences containing regions of homology to a query rbosm or RBOSM of the present invention.

Other approaches to the identification, assembly, storage, and display of nucleotide and polypeptide sequences are provided in "Relational Database for Storing Biomolecule Information," U.S.S.N. 08/947,845, filed October 9, 1997; "Project-Based Fuh-Length Biomolecular Sequence Database," U.S.S.N. 08/811,758, filed March 6, 1997; and "Relational Database and System for

Storing Information Relating to Biomolecular Sequences," U.S.S.N. 09/034,807, filed March 4, 1998, ah of which are incorporated by reference herein in their entirety.

Protein hierarchies can be assigned to the putative encoded polypeptide based on, e.g., motif, BLAST, or biological analysis. Methods for assigning these hierarchies are described, for example, in "Database System Employing Protein Function Hierarchies for Viewing Biomolecular Sequence Data," U.S.S.N. 08/812,290, filed March 6, 1997, incorporated herein by reference.

Identification of Sequence Variants and Polymorphisms

The method comprise a series of filters to identify isSNPs from other sequencing variants and errors. The filters can be grouped into the fohowing five sets of filters by the order of apphcation in the method:

Preliminary Filters: the main filter in the first group removes the majority of base call enors by requiring a minimum phred quahty score of 15. Additional filters at this stage deal with sequence ahgnment enors as weh as errors resulting from improper trimming of vector sequence, chimeras and sphce junctions.

Advanced Chromatogram Analysis: additional base cah enors are then detected by examining the original chromatogram files in the vicinity of a putative SNP by an automated procedure resulting in a set of SNPs wherein the base cah error rate is reduced to less than 5%.

Clone Error Filters: errors introduced during laboratory processing such as those caused by reverse transcriptase, polymerase or somatic mutation are among the most difficult to distinguish from true SNPs. The Clone Enor filters use statisticahy generated algorithms to identify these sources of error. A smah percentage of actual SNPs wih be discarded at this stage.

Clustering Error Filters: these types of errors result from the incorrect clustering of close homologs, pseudo- genes or from contamination by nonhuman sequences. The filters developed to minimize these clustering enors are also statisticahy based. As above these filters may be reject a fraction of actual SNPs

Fimshing Filters: these filters remove duphcate and redundant SNPs from the generated hst of SNP, and remove SNPs which are from the hypervariable regions of hypervariable genes such as immunoglobulin and T cell receptors.

Pre-processing steps

The sequences must first be trimmed to eliminate vector sequence, contamination and repetitive sequences. Then certain low information content sequences (for example, long runs of a single base, or two or three-base repeats) and repetitive sequences (for example Alu sequences in humans) must be massed (changed to N's) to prevent over-clustering enors. The clustering process then identifies the sets of sequences that are believed to be derived from the same original DNA sequence or gene. The sequences in each cluster or then aligned using a method such as phrap which also defines a consensus sequence. It wih be weh recognized by tliose skihed in the art that there are numerous existing programs for canying out these processes, and the SNP discovery process described herein wih work equahy weh with any of them. In the instant embodiment, the preferred processes are Blocked 1 for trimming and masking, a variety of different algorithms for clustering, and phrap for the ahgnment. It wih be recognized by those skihed in the art that phrap and other ahgnment methods cany out a secondary clustering step which divides clusters into contigs, and cany out a secondary triniming step which defines the end points of the portion of each sequence which participates in the contig. The contigs then maybe searched for the occurrence of SNPs.

Errors in the trimming, clustering and ahgnment processes will cause SNP discovery enors, usuahy false positives (the prediction of SNPs where they do not exist). Additional filters which are . the subject of the invention are designed to recognize and remove these errors by providing the abihty to identify likely enors in the processes and to conect them.

In some instances, it is prefened, as an optional step, to unmask regions of sequences which were masked because of low information content or repetitive sequence) during the clustering process can be unmasked after clustering to ahow discovery of SNPs within these regions.

Identification of Candidate SNP Sequences

The first step in identifying candidate SNP sequences is to redefine the end points of each sequence as the points within the previous end points where a stretch of at least 10 consecutive base calls, containing at least eight base changes, matches the consensus sequence exactly. Sequence trimming enors (both at single sequence stage and at the ahgnment stage contribute to the false positives when foreign sequence (vector, chimera or splice variant) is similar to the real sequence and the true boundary is difficult to determine. This step is a conservative approach to avoid false positives and also filters out lower-quahty sequence that the ends. The reason the length of the match with a consensus is measured in base changes is to avoid low significance matches on repetitive sequence such as polyA.

The next step is an each position of the ahgnment to compare the base cahs of all the ahgned sequences which are between their stall and end positions and which have quahty scores greater than a set threshold, and which have neighboring base cahs which agree with a consensus sequence and where the neighboring base cahs also have a quahty score > the threshold. Preferably the threshold is a phred quahty score greater than or equal to 15. The possibilities are A, C, G, T, and -(deletion).

The next step is a Clone Filter where if there has been more than one base cah for a sequence position, then the clone for each sequence is identified in the sequences corresponding to each clone are compared. If the base cahs for different sequences from the same clone disagree, then ah the sequences for this clone at this base position are removed from consideration.

After ah of these filters, positions for which there is more than one base cah are candidate SNPs. The "wild type" base cah is the one in the consensus sequence and the others are designated candidate SNPs. If the wild type base cah is a deletion, then the SNP is considered to be an insertion at the previous base.

Automated Chromatogram Checking

The next filters require opening of the chromatogram files for the sequences identified as containing candidate SNPs. At each candidate SNP position, the chromatogram data of each sequence passing the Identification Filters is extracted. The first step in this process utihzes a program

ABIdump to translate binary ABI chromatogram files into usable form.

Multiple Base Cah Algorithm filter: the ABI base cahs for each sequence are compared to the phred base cahs. If the base cahs do not agree at the SNP position and the two adjacent flanking positions, then the sequences are removed from consideration. Intensity Filter: if the SNP is a single base change (this step is skipped for insertions and deletions), then the process intensity values for each of four bases at the cah chromatogram location of the candidate SNP base are used to compute a ratio. If we cah the intensity of wild type, "wt", the intensity of the SNP base "snp", the minimum of the other two "min", and the phred quahty of the base cah "Q", then the wild type sequences must have (snp-min) < (wt-min)(Q-17)/37 and Q>=17 to be considered high-quality, and

(snp-ιrιin)<(wt-min)(Q-4)/37 and Q>=15 to be considered a low quahty pass.

The basis for these formula is that if a base is mis- called, then there is likely to be a residual peak for the conect base. The larger the peak for the wild type base, the less likely that the cah of the SNP is correct. The actual thresholds in the formula are based on empirical data from clones which were sequence multiple times and which gave a set of confirmed SNPs and error rates for algorithm optimization.

The candidate SNP passes only if at least one wild type sequence passes and at least one SNP sequence passes. The quahty of Hie candidate SNP is the lower of the highest wild type pass level and the highest SNP pass level (if there is a high-quality wild type sequence but only low quahty SNP sequences, then the candidate is low quahty. A SNP quahty value is returned.

Clone Eπor Quahty Filters (somatic mutation/reverse transcriptase/polymerase enors) The purpose of these filters is to remove errors which are actually in the clone, that is, the clone sequence was correct but the clone does not represent the individual being sequenced. Three possible sources of these enors are somatic mutations, enors made by reverse transcriptase in the process of making cDNA, and DNA polymerase errors in those situations where the DNA has been amplified by PCR at some point prior to inserting in the cloning vector. Somatic mutations can be a particular problem in sequencing clones derived from ceh lines.

Polymerase enors are specific to the type of sequencing protocol used. For example, reverse transcriptase is involved in EST sequencing but not genomic clone sequencing. Polymerase is involved in the creation of extension clones (polymerase is used in ah sequencing reactions, but errors are less likely to arise because only a fraction of the templates are affected in contrast to the extension process where a single polymerase product becomes a template for the entire reaction)! This filter is not apphed to genomic sequences in the cunent embodiment on the premise that the genomic sequences do not have polymerase enors, and that somatic mutations are likely to have the same profile as real SNPs.

This filter also filters out rare SNPs as weh as apparent SNPs which are not real. It is difficult to determine and confirm by experiments to what extent SNP candidates are too rare to be confirmed vs. simply not real. For many apphcations, very rare SNPs are of less utility than common ones such that this is not a problem; however in some apphcations it may be advisable to turn this filter off.

Base change sequence analysis filter

The premise of this filter is that probabilities of different mutations is different depending on the source. For example true SNPs may be mostly transitions whereas reverse transcriptase mutations could be primarily G to T mutations. While this does not ahow one to determine for sure that a given change is a true SNP, it allows one to evaluate the relative likelihood that a given mutation is a true SNP. SNP confirmation data suggest that G/T SNP candidates in which there is only one clone having the T ahele have a very low probabihty of being real SNPs. The SNP candidates are excluded from the high confidence set (they are kept in a different file-their confirmation rate is well below 50 percent). The other set which had a very low confirmation rate is any A/T SNP.

Frequency Filter

This filter is based on the concept that true SNPs have a different frequency profile than clone enors and that a candidate SNP which is evident in only one clone in a deep ahgnment is less likely to be real than one which appears in one clone in a shahow ahgnment. The likelihood of finding a SNP at a given sequence location is a function of the number of chromosomes sequenced. This curve is distinctly non-linear as most SNPs are sufficiently frequent, to be found with relatively few sequences. The probabihty of an enor of this type, however is essentiahy linear in the number of sequences since the chance of the change occurring in two different sequences is independent. This means that the probabihty that a candidate SNP observed in a single clone is a true SNP is lower if the ahgnment is deep then if a is shahow. Any SNP occuπing in a single clone in an ahgnment of more than 20 clones (counting only high-quality sequences which have a chance of contributing a candidate ,

SNP) is excluded from the high confidence set.

This filter is the basis of a secondary method used to develop the base change sequence analysis filter. Comparing the set of single clone SNPs from shahow ahgnment's with those from deep ahgnment's, which are more likely to be enors, wih reveal base changes which are more hkely to be associated with polymerase enors and somatic mutations.

Clustering Error Filters These filters are intended to remove candidates SNPs which result from the incoπect clustering of similar sequences such as highly homogenous genes, similar genomic sequences, and contamination from other species where the sequences of the species have been mis- labeled as human.

Number of base change filter

This filter distinguishes homologous sequences from SNPs on the basis of the frequency of variants. True SNPs occur about one per kd when comparing to sequences or once per 2 kb if the length of sequences is included, and this fraction decreases as the depth of the ahgnment increases. Since EST sequences tend to be about 500 bp or less in length, then it would be expected to have not more than one SNP per four sequences. The number of SNPs in the cluster is divided by the number of sequences in the cluster and SNPs for which this number is larger than one are discarded. The higher the number, the less hkely the SNP is to be real. The threshold value of one was chosen because it appears to correspond to roughly a 50 percent success rate, however the threshold value could be adjusted to higher value to accept lower confidence SNPs.

Distance from next polymorphism filter

This filter calculates the number of SNPs for which the sequence is the only representative within a window of 100 bases on either side, and discards any of the SNPs for which there are more than one other SNP in this window. This threshold can be set higher, but the actual fraction of SNP candidates which are true SNPs drops off to less than 50 percent.

Haplotvpe clustering filter When sequences from different sources are inappropriately clustered, it is possible to divide them into two or more clusters which are consistent. In particular, if we take any two differences ' between homologs and consider the haplotypes of the clones which overlap both SNPs, there are only, two haplotypes. In other words, a 2x2 matrix of haplotypes is diagonal having only two non-zero entries. If there are only two sequences, then this is expected. For each SNP, a 2x2 haplotype matrix - with each other SNP is computed. If it is diagonal, and there are more than two sequences, than the sum of the diagonal elements minus one is a "cluster total" for this SNP. This "cluster total" number has proven to be empirically conelated with the confirmation rate, probably because it predicts clusters which contain para-logs, homologs and contamination from other species. Candidates SNPs which have a cluster number of less than eight are kept. This threshold value for the cluster total can be varied.

Redundancy/fMshing filters

Redundant SNP filter: SNPs in different contigs of the same gene which have the same base change and surrounding sequence are flagged as redundant. To accommodate possible splice variants this redundancy filter also apphes to SNPs which have the sunounding sequence matches on only one side.

T ceh receptor/immunoglobulin filters Sequences containing SNPs are filtered to remove SNPs in sequences that are homologs to T ceh receptors and immunoglobulin genes because both types of genes have hyper-variable regions which could result in false positives.

Output file

SNP related data: With each candidate SNP a variety of data is kept, including the number and sources of ah contributing sequences (for example gene album, HTPS, FL, WashU/Merck, etc.), the surrounding sequence, measures of the ratio and quahty scores for the "best" sequence representing each ahele, etc.

Sequence related data: for each sequence associated with each SNP, the fohowing data is kept including the distance in each direction to the end of the sequence, the distance in each direction to the next base different from the consensus and passing the initial quahty filters, the hbrary, tissue ID, donor ID and comments (for example tumor, diseases, normal).

These methods have been described in patent apphcations entitled "Method for the Identification of Sequence Polymorphisms using Polynucleotide Sequence Databases, and Single Nucleotide Polymorphisms Identified Thereby" (Attorney Docket Nos. GX-0006 P and GX-0010 P), and are hereby incorporated by reference.

b. Identification of polymorphisms in osteoarthritis associated genes by SSCP The invention provides methods for detecting the presence of polymorphisms in candidate genes of the invention. The invention also provides methods for distinguishing polymorphisms which contribute to a particular disease (e.g. osteoarthritis) over polymorphisms which do not contribute to the disease.

1. Identification of Polymorphisms in Candidate Genes

Identification of polymorphisms in a candidate gene, according to the invention, wih involve the steps of isolating the candidate gene, deterrrdning its genomic structure and identifying polymorphisms in the DNA sequences in any portion of the entire protein-coding region. The invention also provides methods for identifying polymorphisms in the DNA sequences corresponding to RNA sphce junctions. The invention also provides methods for identifying polymorphisms in the DNA sequence conesponding to the regulatory (promoter) region of the candidate gene. A candidate gene is isolated by cloning methods weh known in the art (described above). Preferably the genomic structure of a candidate gene is determined by Southern blot analysis, as described in Section C. It is expected that the entire sequence of an open reading frame (ORF) of an average entire gene can be spanned by 16 PCR-amphfied DNA fragments or amphmers of an average length of 225 bp. It is expected that a smaher gene can be spanned by 1-2 amphmers and that >50 amphmers are required to span extremely large genes. Primers useful for production of the amphmers of a particular candidate gene are designed based on preexisting knowledge of the sequence of the wild type gene, according to the primer design strategies described in Section A entitled "Design and Synthesis of Ohgonucleotide Primers." For PCR amphfication of a region to be tested by SSCP it is preferable to design primers that amplify overlapping regions of the candidate gene. If a sequence variation is located in a region of a candidate gene that corresponds to the region to which the primers hybridize, the primers wih likely not bind, the region containing this sequence variation wih not be amplified and the variation wih not be detected in PCR based assays. By producing overlapping amphmers it is expected that virtually ah of the sequence variations in a particular candidate gene wih be detected. The amount of overlap in the amphmers is somewhat variable (approximately 20%) and the precise location of the overlapping regions wi depend on the location of regions comprising a sequence that is an appropriate primer : sequence. It is a possibility that a polymorphism wih be located at a position just adjacent to the primer site. Consequently, sequence information wih be available for only 20 bp on one side of the polymorphism and for 104-279 bp on the other side of the polymorphism. However, this should be a sufficient amount of sequence information to ahow definition of a unique sequence context in which to define the particular polymorphism.

Based on screening analysis of 92 samples (184 chromosomes), it is expected that about 50% of the amphmers wih demonstrate polymorphisms, and that approximately 80% of these amphmers wih detect changes at single positions while the remaining 20% wih detect base changes at two positions. Based on these estimates, it is expected that there wih be approximately 10 sequence variations per open reading frame. However, the number of amphmers that demonstrate polymorphisms with vary depending on the number of individuals tested, the ethnicity and structure of the population being tested, and the region of DNA being tested. Preferably, each polymorphism wih be detected in the context of an SSCP fragment.

Polymorphism analysis by fluorescent SSCP (fSSCP, described in detail in Section F entitled "Identification and Characterization of Polymorphisms") uses PCR to generate an amphmer of DNA to be studied. The region to be tested is defined as the region between the primers (e.g. the region that is incorporated into the PCR product and reflects the sequence of the DNA sample being tested). The PCR primers reflect the sequence of the DNA sample being tested and are incorporated into the PCR product as one end of each strand of DNA in the PCR product. If a polymorphism occurs in a primer binding site either the PCR primer does not bind due to the mismatch and the PCR wih not produce a product, or the primer binds, an amphfication step occurs wherein the primer is incorporated, but the amplified product does not contain the polymorphism which occurs at the primer binding site. Therefore, fSSCP provides a method of screening a DNA sequence located between PCR primers for the presence of polymorphisms.

The sensitivity of the technique of fSSCP for detecting a polymorphism is affected by length, such that there is a substantial decrease in the detection of polymorphisms in amphmers that are greater than 300 bp in length. However, different conditions for performing SCCP at high sensitivity with larger fragments, e.g. 800-1500 bp have also been described. If the length of DNA screened per amphmer is decreased then more amphmers are required to screen a region of a given size. Therefore, efficient screening of a gene dictates that the lower limit of the size of an amphmer is 125 bp. To attain specificity for a particular gene sequence, pnmers are usuahy 20-25 bp in length, and additional criteria such as G:C content, and intra- and mter-primer complementarity are important considerations in primer design (as described above). Ah of these considerations are addressed if the primer3 program (Copyright (c) 1996 Whitehead Institute for Biomedical Research) is employed to design pahs of primers suitable for use in a single PCR reaction. Typically, program parameters are set so that multiple amphmers are designed in the length range of 150-300bp, with predicted primer melting temperatures in the naπow range 60-62°C. The nanow temperature range increases the likelihood that a single set of PCR conditions can be used to generate a wide variety of different amphmers. If it is desirable to screen a contiguous stretch of DNA which is larger than the maximum fragment size deshed for sensitive polymorphism detection by fSSCP (300 bp) it is necessary to use multiple amphmers (which are assayed separately) which span the region of interest. Since the primer sites in an amphmer are not tested, these sequences need to be contained within another amphmer. To test the primer sequence, overlapping amphmers are designed by an algorithm that evaluates a large number of amphmers generated by the primer3 program for the optimum overlapping set according to a cost function. Thus, a series of overlapping PCR amphfication products can be used to test a contiguous stretch of DNA. Constraints on primer design are such that the absolute minimum overlap is rarely possible. As a result, some regions of overlap occur that results in 'double testing' of a particular segment of DNA. The detection efficiency is affected by the sequence context of the polymorphism; it is possible that a polymorphic site wih be detected in only one of two different amphmers which overlap the same site. One strategy that is useful for increasing polymorphism detection efficiency is to design overlapping amphmers to generate 2-fold coverage of ah sequences.

SSCP does not detect 100% of polymorphisms. The invention provides for detection of polymorphisms with an efficiency of 95% under a single set of conditions using single coverage of sequences; a 2-fold screening strategy can be employed if it is necessary to increase this detection efficiency.

It is expected that the polymorphism can be located, and detected anywhere in the SSCP fragment except in the regions at each end that correspond to the sequence of the PCR primers. The precise location and identity of the sequence variations) of a particular SSCP fragment can be confirmed by sequencing the fragment as described in Section D entitled "Isolation of a Wild Type Gene". The sequence of a candidate gene wih be compared to the known sequence of a wild-type version of the gene by using the fohowing DNA/protein sequence analysis programs and methods.

There are a large number of freely available methods for perfoiming sequence comparisons. These methods differ in their speed of execution, their sensitivity, and the type of comparisons they are able to make. For example one can compare two DNA sequences, two protein sequences, a DNA sequence to a protein sequence by conceptual translation, or DNA sequences as if they were protein sequences, again by conceptual translation. The. BLAST suite of programs (Altschul et al, 1990, J.MolBiol 215:403) are commonly used to perform the above-referenced type of analysis. Although the BLAST suite of programs provides a rapid method of deterrriining multiple distinct similarities between two sequences, these programs are not guaranteed to find an optimal solution when comparing two sequences according to a particular set of parameters. PSI-BLAST is a more sensitive variant of BLAST that operates by iteratively searching the database while simultaneously refining the query pattern based on the results of the searches. Other packages of programs that are available and which have different specific properties include the HMMER, SAM, WISE, STADEN and FASTA packages, and the programs est_genome, dotter, e-PCR, Clustal, crossjmatch and phrap (Pearson, 1996, Methods Enzymol. 266:227).

If sequence information is available for the intron-exon boundaries and for a region of the intron (of approximately 30-150 bp) located immediately 5' of an intron-exon boundary, primers can be designed to produce amphmers useful for identifying polymorphisms located in the RNA splice junctions. Similarly, if the promoter region of a candidate gene has been sequenced, primers can be designed to produce amphmers useful for identifying polymorphisms located in the promoter region. Additional methods for detecting and isolating polymorphisms include, but are not limited to fluorescent polarization-TDI, mass spectroscopy denaturing gradient gel electrophoresis, chemical cleavage of mismatch, constant denaturant capillary electrophoresis, RNase cleavage, heteroduplex analysis, sequencing by hybridization, DNA sequencing, representational difference analysis, and denaturing high performance hquid chromatography, described below in Section F entitled, "Identification and Characterization of Polymorphisms".

2. Methods of Determining if a Polymorphism Contributes to osteoarthritis No two individuals (excluding identical twins or other clones) have the same sequence of DNA in their genome. Variability in gene sequences between individuals accounts for many of the obvious phenotypic differences (such as pigmentation of hah, skin, etc.) and many nonobvious ones (such as drug tolerance and disease susceptibihty). In a population, the DNA sequence that occurs at the highest frequency at any given site is commonly referred to as the wild type sequence. The term "wild type sequence" can be misleading, however, because in different populations an alternative form of a DNA sequence maybe predominant and thus considered wild type for that particular population. DNA polymorphisms are located throughout the genome, within and between genes, and the various forms may or may not result in differential gene function (as determined by comparing the function of two alternative forms of the same sequence). Most polymorphisms do not alter gene function and are cahed neutral polymorphisms. Some polymorphisms do have an effect on gene function, for example - by changing the amino acid sequence of a protein, or by altering control sequences such as promoters or RNA splicing or degradation signals. Polymorphisms can be used in genetic studies to identify a gene involved in a disease. If a polymorphism alters a gene function such that it increases disease susceptibihty, then it will be present more often in individuals with the disease than in those without the disease. Alternatively, if a particular DNA variant is protective against a disease, it wih be found more often in individuals without the disease than in those with the disease. Statistical methods are used to evaluate polymorphism frequencies found in diseased as compared to normal populations, and provide a means for estabhshing a causal link between a polymorphism and a phenotype. To detect a significant association between a disease and a polymorphic site, different tests maybe used with either genotypic or ahelic distributions. The simplest test consists of a t-test wherein the frequency of the polymorphic aheles in normal individuals and individuals with the disease phenotype is compared. A comparison of the genotypic distribution in normal individuals and individuals with the disease phenotype can also be performed using a chi-square test of homogeneity. These tests are implemented in ah commerciahy or freely available statistical packages, for example SAS and S+, and are even included in Microsoft Excel. More sophisticated analyses wih be performed by incorporating covariates such as linear regression or logistic regression, and by accounting for the information provided by adjacent polymorphic sites (multipoint analysis). An example of this type of program is the freely available program "Analyze" by JD Terwilhger (currently available at the WWW site ftp://ftp.weh.ox.ac.uk/pub/genetics/analyze). If a polymorphism has a phenotypic effect, a bias wih exist in the distribution of polymorphisms between groups that have and do not have the disease phenotype. This manner of analysis can be used to study a trait that is not necessarily a disease; any trait can be studied by comparing a group with a particular phenotypic form of a trait to a group with a different phenotypic form of that trait. It is important that the cases and controls are correctly matched with regards to ethnicity, envhonmental influences, and other factors which could effect the phenotype being studied. Studies which test polymorphism frequencies within groups exhibiting different phenotypes and use statistical methods to compare the group polymorphism frequencies and identify correlations with phenotypes, are known as "associations studies".

Some polymorphisms that occur in a single gene can alter the function of a gene sufficiently such that the polymorphism results in a disease (monogenic disease). However, many common human diseases are polygenic; that is they are the result of complex interactions of various forms of multiple genes. In the case of polygenic diseases, the alteration of a single gene may not be detrimental per se, but in combination with certain sequence variants of other genes, this altered DNA sequence may contribute to a disease phenotype. DNA variants leading to monogenic diseases are usually rare in a population due to the process of natural selection against tliose caπying the disease gene. As variants in genes that are involved in polygenic disease do not produce the disease phenotype unless they occur in the appropriate combination with other gene variants, normal individuals can cany a subset of the disease-contributing variants without suffering adverse effects. Thus, disease-contributing gene variants that are associated with polygenic diseases may exist at a high frequency in a normal population. Selection against these disease variant forms of a gene wih only occur when they are present in the appropriate disease-causing combination and there may not necessarily be selection against these gene variants in individuals caπying a subset of the disease-contributing variants. Neutral DNA variants do not alter gene function or contribute to a disease, are under no selective pressure and occur at variable frequencies wifliin populations.

Monogenic diseases tend to be rare wifliin the population, and therefore few patients maybe available for studies of these diseases. A polymorphism in a single specific gene is necessary and usually sufficient to cause a monogenic disease, such that associations between the variant gene and the phenotype are usuahy readily apparent. In cases where the expression of a mutation phenotype is complete, ("complete penetrance"), the polymorphism present in the disease gene wih not be found upon examination of a large number of normal individuals. If there is not complete penetrance then some apparently normal individuals wih contain the mutation; the difference in frequency of occurrence of the variant gene in the disease group as compared to the normal population will reveal that the variant is associated with the disease. In polygenic diseases, variation at different genes occurs in a combination which alters susceptibihty to the disease. Although several genes may have variant forms which can contribute to a disease phenotype, it is not always necessary for a contributing variant to be present at every gene potentiahy contributing to the disease in a given affected individual. For example, a hypothetical disease could be caused by a particular combination of variants at three of four genes, designated as A, B, C, and D. Appropriate susceptibihty variants in combination at any three of the genes can cause the susceptibihty, i.e. one person with increased susceptibihty may have susceptibihty variants in genes A, B, and C, while another individual with increased susceptibihty to the same disease wih have susceptibihty variants in genes B, C, and D. Therefore, although not ah affected individuals wih have the same susceptibihty variants, the net result is that a diseased population wih have susceptibihty variant forms of genes A, B, C, and D at a higher frequency than an unaffected population (as detected by association studies).

Unlike monogenic diseases which result from polymorphisms that are not present in control " populations, the polymorphisms which contribute to the polygenic disease are also present in a normal population. As described in the example above, an individual with susceptibihty polymorphisms in only one or two of the genes potentiahy contributing to the disease susceptibihty wih be normal with regard to disease susceptibihty. Therefore, normal populations can be used to identify polymorphic regions of the genome in the population, and these regions can then be specifically tested in larger patient and control populations. Typically, a gene is analyzed for the presence of polymorphisms by testing between 2 and 100 normal individuals in order to estabhsh if a particular polymorphism is present for that gene in the population. Once a polymorphic site(s) has been defined, the polymorphic site is then tested in case (disease) and control (normal) populations and statistical analyses are performed to identify polymorphisms which occur at significantly different frequencies in the two populations. The determination of the statistical significance of polymorphism frequency differences is dependent upon the size of the observed frequency difference between the populations, and on the size of the populations being studied. If a significant difference is found, then it can be concluded that an association exists between the polymorphism and the phenotype being studied. A statisticahy significant difference is a frequency difference at a particular site between populations which would be expected to occur by chance in only 5 out of 100 tests. That is, a difference which has a 95% probabihty of being a true difference due to the affect of the gene.

The foregoing discussion describes a method of testing for an association between a polymorphism which is the direct contributor to a disease and the disease phenotype. However, polymorphisms which do not directly contribute to a disease can also be used to identify regions of the genome which contain genes that contribute to the disease by virtue of their proximity to disease- contributing polymorphisms.

In humans, DNA exists as 23 homologous pairs of linear molecules (chromosomes). Recombination is a process which results in reciprocal exchanges of short homologous DNA segments between tliese homologous DNA pahs. Only one of each of the 23 pahs of chromosomes is inherited by the offspring. The inherited chromosome is thus made up of tandemly arrayed segments of DNA derived from both of a pah of chromosomes. Consequently, DNA is transferred in segments from one generation to the next. Although the boundaries of each inherited segment may vary in each generation, the net effect is that sequences of DNA which are adjacent along the length of the molecule are inherited together at a higher frequency than sequences that are farther apart. If a region (continuous linear segment) of DNA has two or more polymorphisms that are close together, they wih be co-inherited at a higher frequency than polymorphisms that are farther apart, as they are more hkely to remain on the same segment of DNA during recombination. Therefore, if two or more polymorphisms are close together, they wih occur together at a higher frequency in a population than would be expected by random segregation. This effect is known as linkage. Linkage studies are performed using multiply affected individuals within famihes; the most commonly used approach is to test markers located throughout the genome in many sets of affected sib pahs that share the same phenotype. Markers which are located in the region of a genome that contributes to the phenotype wih be inherited in both siblings, along with the phenotype, at a higher frequency than expected by chance. Studies wherein data from many such famihes is compared can be used to implicate a region of a genome as one that contributes to a particular phenotype.

Linkage disequihbrium (LD) association studies provide another method for using polymorphisms in genetic studies. The method of LD involves making a correlation at the population level, between the aheles (alternative polymorphic forms of the same sequence site) present at different genomic sites. If site 1 has two variant forms, A and a, and site 2 has two variant forms B and b, the observation in a population that ahele A at site 1 is more often found with ahele B at locus 2 than with ahele b is an example of LD. If ahele B is a disease- contributing polymorphism, then testing at ahele A may show an association with the disease.

Linkage disequihbrium maybe generated in several ways. Maintenance of LD in a population allows a disease association to be detected many generations after the formation of LD. The maintenance of LD is explained by linkage: the closer the two loci, the longer (in terms of number of generations) that particular LD is maintained. As a result, polymorphisms which do not directly contribute to a disease can be used to identify regions of the genome which contain a disease contributing polymorphism. If a polymorphism affects gene function such that it contributes to a phenotype being studied and is found to be associated with the phenotype, nearby (neutral) polymorphisms which are in LD with the disease polymorphism may also show an association with the disease. Conversely, if a polymorphism does not affect gene function but is found to be associated with a particular phenotype, this polymorphism is in LD with a different, but adjacent polymorphism that affects gene function such that it contributes to the phenotype being studied. If a neutral polymorphism is always inherited with a phenotype- contributing polymorphism, then the strength of the association of the neutral polymorphism to the phenotype wih be equal to that of the polymorphism which affects gene function and is contributing to the phenotype. A polymorphism which shows an association with a phenotype (for instance with disease susceptibihty) is a marker for that phenotype and imphcates the region in which the polymorphism resides as a region containing a polymorphism which contributes to the phenotype. Additional flanking polymorphisms can be tested to determine the precise location of the true phenotype-contributing variant.

Linkage studies on famihes, and LD studies on populations have different degrees of resolution with regards to defining the size of a DNA region which contains the phenotype- contributing polymorphism. In general, linkage studies define an interval which potentiahy contains tens to hundreds of genes, while LD studies have been used to implicate single genes in the development of a particular phenotype.

3. Test Populations Useful for Polymorphism Genotyping The invention provides methods of determining ahehc frequencies by performing genotypic analyses in appropriate test populations. Study cohorts:

Osteoarthritis Progression Cohort Derived from a population of normal women aged 45-65. The original aims of the study, started in 1989, were to assess how many women around menopausal age would get arthritis and what factors predispose them to developing it. Also to lookinto factors that may be associated with progression of the disease. A series of examinations, x-rays and questionnaires about hfestyle factors were carried out on 1003 women that were recroited to the study. This study has been going for 10 years. As a result, a unique, world-renowned and weh respected study is avaflable looking at the reasons why women develop osteoarthritis, potential risk factors and the genetics of the disease.

Prospective Severe Outcomes Cohort (case-control)

Five hundred joint replacement cases wih be ascertained as wih be age, ethnicity and gender matched controls. The clinical data envisaged are : HRT use, numbers of joints affected, occupation, injury history, age, BMI.

The hst of studies relevant is shown in fohowing table.

HI •MMMM mm

Pilot¹ 100 progressors + 75 non-progressors, Large genetic effects for 6-8 Months

100 normals, all female from the fast OA progression, proof progression, cohort Detailed, clinical of principle. Correlation data, 10 yr. fαllow-up: joint-space with biomarkers. Possible narrowing/yr., joints affected, BMD, novel target fractures, CRP levels.

Biomarker 800 women from progression cohort. Correlation of genetics with 12 Months study DNA, serum, urine, Shiomarkers biomarkers - v, useful for clinical trials,

Progression ~800 women from progression cohort. Genetic effects of OA. - 18 Months . hand & knee Detailed chnical data. joint-space ' progression. Risk of OA. OA study iiarrowing/yr. , joints affected, BMD ' Correlation with biomarkers. (Mp and spine), fractures, CRP levels, Possible novel target. ¹ fufl lipid measurements, incidence of Genetic effects of fractures (assessed by X-rays), 10 yr. osteoporosis risk, coπelation follow-up radiographs for ah patients. with BMD.

Possibly genetic effects of lipid levels and CVD risk.

Case-control ~5P0 cases (joint replacements) Vs 500 Large genetic effects for -6-12 matched controls. Prospective study: OA risk, proof of principle, months for DNA + % biomarkers. Clinical data Possible novel target collection required! steroid use, fjoints affected, 4. Assays Useful for Determining the Association of a Polymorphism with osteoarthritis

Clinical parameters

There is a general consensus that radiological changes are the prefeπed method for epidemiological studies on the basis of cross sectional and prospective conelations between severity of X-ray changes with the presence of pain and loss of function. In osteoarthritis, the loss of cartilage produces a narrowed space between bones. The pattern of joint space narrowing can help distinguish between osteoarthritis and rheumatoid arthritis. Bone spurs (osteophytes) also help diagnose osteoarthritis. Other relevant clinical end points are pain, disability, function, joint replacement and maintenance of joint structure. Stages of disease progression are as fohows:

Early stage: focal swelling of articular cartilage fohowed by the appearance of hregulariti.es in the surface.

Intermediate stage: progressive degradation and loss of articular cartilage. Also characterised by fibrillation (vertical sphtting), detachment (horizontal sphtting) and thinning of the cartilage.

Late stage: Articular cartilage is almost completely destroyed. Bony outgrowths (osteophytes) occur at the joint margins resulting in residual arthritis. Characterised by pain and limitation of joint movement.

Clinical measurements of OA

Quantitative traits of interest for the study of OA and its progression are:

- Osteophyte count.

Joint space nanowing (mm/yr.) Number of joints affected Types of joints affected¹ In addition a series of biochemical markers can provide valuable information such as:

COMP - CRP - HA

Protocollagen Type II

Bone resorption markers (e.g. collagen cross-links)

Confounding factors Most cunently recognised envhonmental risk factors for prevalent knee OA - obesity, knee injury, and physical activity, influence incidence more than radiographic progression. Furthermore, these factors might selectively influence osteophyte formation more than joint space narrowing. These findings are consistent with knee OA being initiated by joint injury, but with progression being a consequence of impaired intrinsic repair capacity. Other known confounding factors are steroid (glucocorticoid) use and, in women, hormone replacement therapy. Glucocorticoids ameliorate erosion in animal OA models and suppress synthesis of matrix metahoproteinases (Saito et al. 1999). Estrogen replacement therapy, on the other hand, has been shown to have a moderate, but not statisticahy significant, protective effect against worsening of OA both in the Chingford (Hart et al. 1999) and Framingham (Zhang et al. 1998) studies.

5. Methods of Genotyping Polymorphisms

The invention discloses methods for performing polymorphism genotyping. These methods can be used to detect the presence of a polymorphism in a sample comprising DNA or RNA.

A DNA sample for analysis according to the invention may be prepared from any tissue or ceh line, and preparative procedures are weh-known in the art. The preparation of genomic DNA is performed as described in Section B.

RNA samples may also be useful for genotyping according to the invention. Isolation of RNA can be performed according to the fohowing methods.

RNA is purified from mammalian tissue according to the fohowing method. Fohowing removal of the tissue of interest, pieces of tissue of <2g are cut and quick frozen in hquid nitrogen, to prevent degradation of RNA. Upon the addition of a volume of 20 ml tissue guanidinium solution per 2 g of tissue, tissue samples are ground in a tissuemizer with two or three 10-second bursts. To prepare tissue guanidhum solution (1 L) 590.8 g guanidinium isothiocyanate is dissolved in approximately 400 ml DEPC-treated H.0. 25 ml of 2 M Tris-Cl, pH 7.5 (0.05 M final) and 20 ml Na^EDTA (0.01 M final) is added, the solution is stirred overnight, the volume is adjusted to 950 ml, and 50 ml 2-ME is added.

Homogenized tissue samples are subjected to centrifugation for 10 min at 12,000 x g at 12°C. The resulting supernatant is incubated for 2 min at 65°C in the presence of 0.1 volume of 20%

Sarkosyl, layered over 9 ml of a 5.7M CsCl solution (O.lg CsCl/ml), and separated by centrifugation overnight at 113,000 x g at 22°C. After careful removal of the supernatant, the tube is inverted and drained. The bottom of the tube (containing the RNA pehet) is placed in a 50 ml plastic tube and incubated overnight (or longer) at 4°C in the presence of 3 ml tissue resuspension buffer (5 mM EDTA, 0.5% (v/v) Sarkosyl, 5% (v/v) 2-ME) to ahow complete resuspension of the RNA pehet. The resulting RNA solution is extracted sequentially with 25:24:1 phenol/chloroform/isoamyl alcohol, fohowed by 24:1 chloroform/isoamyl alcohol, precipitated by the addition of 3 M sodium acetate, pH 5.2, and 2.5 volumes of 100% ethanol, and resuspended in DEPC water (Chirgwin et al, 1979, Biochemistry, 18: 5294). Alternatively, RNA is isolated from mammalian tissue according to the fohowing single step protocol. The tissue of interest is prepared by homogenization in a glass teflon homogenizer in 1 ml denaturing solution (4M guanidhum thiosulfate, 25 mM sodium citrate, pH 7.0, 0.1 M 2-ME, 0.5% (w/v) N-laurylsarkosine) per lOOmg tissue. Fohowing .transfer of the homogenate to a 5-ml polypropylene tube, 0.1 ml of 2 M sodium acetate, pH 4, 1 ml water-saturated phenol, and 0.2 ml of 49:1 chloroform/isoamyl alcohol are added sequentiahy. The sample is mixed after the addition of each component, and incubated for 15 min at 0-4°C after ah components have been added. The sample is separated by centrifugation for 20 min at 10,000 x g, 4°C, precipitated by the addition of 1 ml of 100% isopropanol, incubated for 30 minutes at -20°C and pelleted by centrifugation for 10 minutes at 10,000 x g, 4°C. The resulting RNA pehet is dissolved in 0.3 ml denaturing solution, transfened to a microfuge tube, precipitated by the addition of 0.3 ml of 100% isopropanol for 30 minutes at -20°C, and centrifuged for 10 minutes at 10,000 x g at 4°C. The RNA pehet is washed in 70% ethanol, dried, and resuspended in 100-200 ml DEPC-treated water or DEPC-treated 0.5% SDS (Chomczynski and Sacchi, 1987, Anal. Biochem., 162: 156).

RNA prepared according to either of these methods can be used for genotyping by the methods of Northern blot analysis, SI nuclease analysis and primer extension analysis (Ausubel et al, supra). cDNA samples also maybe prepared according to the invention, i.e., DNA that is complementary to RNA such as mRNA. The preparation of cDNA is weh-known and weh- documented in the prior art. cDNA is prepared according to the fohowing method. Total cellular RNA is isolated (as described) and passed through a column of ohgo(dT)-cehulose to isolate polyA RNA. The bound polyA mRNAs are eluted from the column with a low ionic strength buffer. To produce cDNA molecules, short deoxythymidine ohgonucleotides (12-20 nucleotides) are hybridized to the polyA tails to be used as primers for reverse transcriptase, an enzyme that uses RNA as a template for DNA synthesis. Alternatively, mRNA species can be primed from many positions by using short ohgonucleotide fragments comprising numerous sequences complementary to the mRNA of interest as primers for cDNA synthesis. The resultant RNA-DNA hybrid can be converted to a double stranded DNA molecule by a variety of enzymatic steps weh-known in the art (Watson et al, 1992, Recombinant DNA, 2nd edition, Scientific American Books, New York).

Tissues or fluids which are useful for obtaining a DNA or RNA sample according to the invention include but are not limited to plasma, serum, spinal fluid, lymph fluid, external secretions of the skin, respiratory, intestinal and genitoruinary tracts, sahva, blood cehs, tumors, organs, tissue and samples of in vitro ceh culture constituents.

Genotyping methods which are useful according to the invention, i.e., for the detection of polymorphisms in nucleic acid samples isolated from individuals, are disclosed below. , .

Single Strand Conformation Polymorphism (SSCP) Screening and Fluorescent SSCP Screening (fSSCP)

SSCP Analysis

One technique for detecting DNA sequence variations in a biological sample is single strand conformation polymorphism (SSCP) (Glavac et al, 1993, Hum. Mut. 2:404; Sheffield et al, 1993, Genomics 16:325). SSCP is a simple and effective technique for the detection of single base changes. This technique is based on the principle that single-stranded DNA molecules assume specific sequence-based secondary structures (conformers) under nondenaturing conditions. The detection of point mutations by single stranded conformation polymorphism is beheved to be due to an alteration in the structure of single stranded DNA. Molecules differing by only a single base substitution may assume different conformers and migrate differently in a nondenaturing polyacrylamide gel. Single stranded DNAs that contain sequence variations are identified by an abnormal mobility on polyacrylamide gels. SSCP detects ah types of point mutations and short insertions or deletions that are located between the PCR primers (within the probe region) with apparently equal efficiency. This technique has proven useful for detection of multiple mutations and polymorphisms, including SNPs. SSCP sensitivity varies dramatically with the size of the DNA fragment being analyzed. The optimal size fragment for sensitive detection by SSCP is approximately 125-300bp.

The mobihty of a single stranded DNA or double stranded DNA fragment during electrophoresis through a gel matrix is dependent on its size. Smah molecules migrate more rapidly than large molecules because they pass through the pores in the matrix more easily. Conventionahy, electrophoresis of single stranded DNA involves a 'denaturing' gel which maintains the single strandedness of the molecules. The denaturant is typically urea in polyacrylamide gels, and typically formamide or sodium hydroxide in agarose gels. In contrast, according to the SSCP screening protocol, single-stranded DNA is analyzed on a 'nondenaturing' gel. When single stranded DNA is analyzed on a 'non-denataring' gel, intramolecular interactions can occur. In particular, the single stranded DNA is able to (partially) bind to itself. Consequently, DNA that is separated by electrophoresis on an SSCP gel does not migrate as a linear molecule but rather, the mobihty of the DNA on an SSCP gel is governed by both its size and tertiary structure (conformation). The tertiary structure of a single stranded DNA fragment is dependent on the sequence of the entire fragment.

Therefore, if a polymorphism exists in a given fragment, the conformation wih usually be altered. The technique is performed as fohows. '

One or more test DNA samples are prepared for analysis as described above, and subject to PCR amphfication. Ohgonucleotide primers are designed and synthesized as described above. Amphfications are performed in a total volume of 10 ml containing 50 mM KCl, 10 mM Tris-HCl, pH 9.0 (at 25°C), 0.1 % Triton X-100, 1.5 mM MgCl₂, 0.2mM of dGTP, dATP, dTTP, 0.02 mM of non radioactive dCTP, 0.05 ml [a-³³P] dCTP (1,000-3,000 Ci mmol¹; 10 mCi ml¹), 0.2 uM each primer, 50 ng genomic DNA (or 1 ng of cloned DNA template) and 0.1 U Taq DNA polymerase. The PCR cycling profile is as fohows : preheating to 94°C for 3 min fohowed by 94°C, 1 min; annealing temperature, 30 sec; 72°C, 45 sec for 35 cycles and a final extension at 72°C for 5 min. Annealing temperature is different for each PCR primer pah and can be optimized according to the parameters described above. Amphfications using Vent Taq polymerase (New England Biolabs) are performed in a total volume of 10 ul using the buffer provided by the manufacturer with 1 mM each of dGTP, dATP, dTTP, 0.02 mM dCTP, 0.25 ul [a-³³P] dCTP (1,000-3,000 Ci mmol^lO mCi ml¹), 0.2 uM of each primer, 50 ng of genomic DNA (or 1 ng of cloned DNA template) and 0.1 U of Vent Taq DNA polymerase. Samples are heated to 98°C for 5 min prior to addition of enzyme and nucleotides. The PCR cycling profile is 98°C, 1 min; annealing temperature, 45 sec; 72°C, 1 min for 35 cycles, fohowed by a final extension at 72°C for 5 min. The length and temperature of each step of a PCR cycle, as weh as the number of cycles, is adjusted in accordance to the stringency requirements, as described above.

SSCP analysis is performed as fohows. Ten ul of formamide dye (95% formamide, 20mM

EDTA, 0.05% bromophenolblue, 0.05% xylene cyanol) are added to 10 ul ahquots of radiolabeled PCR product. Fohowing denaturation at 100°C for 5 min, the reaction mixture is placed on ice. Two ul ahquots are loaded onto 8% acrylamide:bisacrylamide (37.5:1), 0.5X TBE (45 mM Tris-borate, 1 mM

EDTA), 5% glycerol gels. Electrophoresis is caπied out at 25W at 4°C for 8 hours in 0.5X TBE.

Dried gels are exposed to X-OMAT ARfihn (Kodak) and the autoradiographs are analyzed and scored for aberrant migration of bands (band shifts). SSCP maybe optimized, as deshed, as taught in Glavac et al, 1993, Hum. Mut. 2:404.

fSSCP Analysis

Techniques for screening multiple DNA samples simultaneously are also useful for performing rapid genotyping analysis on a large number of samples according to the invention. By pooling and multiplexing DNA samples in fluorescent SSCP (fSSCP) assays, the high throughput required for detecting sequence variations in a large number of samples is achieved (Makino et al, 1992, PCR Methods Appl. 2:10; Ellison et al., 1993, BioTechniques 15:684). According to the method of fSCCP, PCR products are visualized and analyzed using an ABI fluorescent DNA sequencing machine. Different primer pahs are identified by different color fluorochromes (4 different fluorochromes are now available). fSSCP offers the fohowing advantages over SSCP. Unlike SSCP, fSSCP does not require handling of radioactive materials. Furthermore, the fSSCP technique ahows for automated data and automated data analysis programs that detect aberrantly migrating samples. In contrast, SSCP evaluation involves visual examination by an individual, and does not provide a means for coπecting for lane to lane variations in electrophoretic conditions, as does fSSCP analysis. fSSCP Analysis is performed as fohows.

Amphfications are performed in a total volume of 10 ul containing 50 mM KCl, lOmM Tris- HCl, pH 9.0 (at 25 °C), 0.1 % Triton X-100, 1.5 mM MgCl^, 0.2mM of dGTP, dATP, dTTP, dCTP, 0.2 uM primer labeled with one of the fluorochromes HEX, FAM, TET or JOE, 50 ng genomic DNA (or 1 ng of cloned DNA template) and 0.1 U Taq DNA polymerase. The PCR cycling profile is as fohows : preheating to 94°C for 3 min fohowed by 94°C, 1 min; annealing temperature, 30 sec; 72°C, 45 sec for 35 cycles and a final extension at 72'C for 5 min. Annealing temperature is different for each PCR primer pah. Amphfications using Vent Taq polymerase (New England Biolabs) are performed in a total volume of 10 ul using the buffer provided by the manufacturer with 1 mM each of dGTP, dATP, dTTP, dCTP, 0.2 uM primer labeled with one of the fluorochromes HEX, FAM, TET or JOE, 50 ng genomic DNA (or 1 ng of cloned DNA template) and 0.1 U of Vent Taq DNA polymerase. Samples are heated to 98°C for 5 min prior to addition of enzyme and nucleotides. The PCR cycling profile is 98°C, 1 min; annealing temperature, 45 sec; 72°C, 1 min for 35 cycles, followed by a final extension at 72°C for 5 min. Anneahng temperatare is different for each PCR primer pah. Two ul of fluorescent PCR products are added to 3 ul foimamide dye (95% formamide, 20mM EDTA, 0.05% bromophenolblue, 0.05% xylene cyanol), denatured at 100°C for 5 min, then placed on ice. Thereafter, 0.5-1 ml of Genescan™ 1500 size markers are added as an internal standard. Two ul of the mix is loaded onto 8% or 10% acrylamide:bisacrylamide (37.5:1), 0.5X TBE (45 mM Tris- borate, 1 mM EDTA), 5% glycerol gels and electrophoresis is performed on an ABI 377 DNA sequencing machine. Gel temperature is maintained between 4° and 10°C by an external cooling unit connected to the internal cooling plumbing and chambers. Electrophoresis is carried out at 2500-3500 volts for 4 - 10 hours in 0.5X TBE. Data is automatically collected and analyzed with Genescan and Genotype analysis software (ABI). The fSSCP procedure identifies regions of 150-300 base pahs containing a sequence variation. To identify the exact sequence change, the fragment which demonstrates the abeπant migration is amplified again from the same biological sample, using non fluorescent primers. The ^: sequence is then determined using standard DNA sequencing methods weh known to those skihed in the art (Ausubel et al, supra). Although SSCP and fSSCP techniques are prefeπed according to the invention, other methods for detecting sequence variations, including DNA sequencing, can be employed. Additional techniques for detecting DNA sequence variations useful according to the invention are described below.

Fluorescence Polarization-TDI

Fluorescence polarization-TDI is another prefeπed technique technique according to the invention for the detection of sequence variations. Template-directed primer extension is a dideoxy chain terminating DNA sequencing protocol designed to ascertain the nature of the one base immediately 3' to the sequencing primer that is annealed to the target DNA immediately upstream from the polymorphic site. In the presence of DNA polymerase and the appropriate dideoxyribonucleoside triphosphate (ddNTP), the primer is extended specifically by one base as dictated by the target DNA sequence at the polymorphic site. By deteπnining which ddNTP is incorporated, the aheles present in the target DNA can be determined. Fluorescence polarization is based on the observation that when a fluorescent molecule is exited by plane-polarized hght, it emits polarized fluorescent hght into a fixed plane if the molecules remain stationary between excitation and emission. However, because the molecule rotates and tumbles in solution, fluorescence polarization is not observed fully by an external detector. The fluorescence polarization of a molecule is proportional to the molecule's rotational. relaxation time, which is related to the viscosity of the solvent, absolute temperature, molecular volume, and the gas constant. If the viscosity and temperature are held constant, then fluorescence polarization is directly proportional to the molecular volume, which is directly proportional to the molecular weight. If the fluorescent molecule is large (with high molecular weight), it rotates and tumbles more slowly in solution and flourescence polarization is preserved. If the molecule is smah (with low molecular weight), it rotates and tumbles faster and fluorescence polarization is largely lost (depolarized).

In the FP-TDI assay, the sequencing primer is an unmodified primer wih its 3' end immediately upstream from a polymorphic or mutation site. When incubated in the presence of ddNTPs labled with different fluorophores, the ahele-specific dye ddNTP is incorporated onto the TDI primer in the presence of DNA polymerase and target DNA. The genotype of the target DNA molecule can be determined simply by exciting the fluorescent dye in the reaction and determining whether a change in fluorescence polarization occurs.- Chen et al, 1999, Genome Res., 9:492.

One or more test DNA samples are prepared for analysis as described above, and subject to PCR amphfication. Ohgonucleotide primers are designed and synthesized as described above. Amphfications are performed in a total volume of 10 ml containing 50 mM KCl, 10 mM Tris-HCl, pH 9.0 (at 25°C), 0.1 % Triton X-100, 1.5 mM MgCl., 0.2mM of dGTP, dATP, dTTP, 0.02 mM of non radioactive dCTP, 0.05 ml [a-³³P] dCTP (1,000-3,000 Ci mmol¹; 10 mCi ml¹), 0.2 uM each primer, 50 ng genomic DNA (or 1 ng of cloned DNA template) and 0.1 U Taq DNA polymerase. The PCR cycling profile is as fohows : preheating to 94°C for 3 min fohowed by 94°C, 1 min; anneahng temperature, 30 sec; 72°C, 45 sec for 35 cycles and a final extension at 72°C for 5 min. Annealing temperature is different for each PCR primer pah and can be optimized according to the parameters described above. Amphfications using Vent Taq polymerase (New England Biolabs) are performed in a total volume of 10 ul using the buffer provided by the manufacturer with 1 mM each of dGTP, dATP, dTTP, 0.02 mM dCTP, 0.25 ul [a-³³P] dCTP (1,000-3,000 Ci mmolMO mCi ml¹), 0.2 uM of each primer, 50 ng of genomic DNA (or 1 ng of cloned DNA template) and 0.1 U of Vent Taq DNA polymerase. Samples are heated to 98°C for 5 min prior to addition of enzyme and nucleotides. The PCR cycling profile is 98°C, 1 min; annealing temperature, 45 sec; 72°C, 1 min for 35 cycles, fohowed by a final extension at 72°C for 5 min. The length and temperature of each step of a PCR cycle, as well as the number of cycles, is adjusted in accordance to the stringency requirements, as described above.

Fohowing PCR amphfication, unused PCR primers and dNTPs are destroyed by adding 2ml of PCR product to 2ml of SAP/Exonuclease cocktail (0.1U shimp alkaline phosphatase (1 U/ml,Amersham Pharmacia Biotech, Inc., Piscataway, NJ)and 0.2U E. coli exonuclease I (10 U/ml, Amersham)hι SAP buffer (20mM TrisHCl, pH 8.0; 10 mM MgCl₂, Amersham))per weh of a 384-weh Black PCR plate (ABT). The mixtures are incubated at 37°C for 60 min before the enzymes are heat inactivated at 95°C for 15 min. The mixture is held at 4°C until used in the FP-TDI assay.

To the enzymaticahy treated PCR product, 2 ml of TDI reaction cocktail containing TDI buffer (50mM Tris-HCl (pH 9.0), 50mM KCl, 5 mM NaCl, 2 mM MgCl., 8% glycerol), 1 mM TDI primer, 12.5 nM of each of two ahele specific dye-labled ddNTPs (ROX-ddGTP, BFL-ddATP, Tamra-ddCTP, or R6G-ddUTP; NEN Life Science Products, Inc., Boston, MA), and 0.32U Thermo Sequenase (Amersham). The reaction mixtures are incubated at 94oC for 15 min, fohowed by 34 cycles of 94°C for 30 seconds and 55°C for 15 seconds. Upon completion of the reaction cycles, the samples are held at 4°C.

After the primer extension reaction, 24 ml of TE buffer/methanol (2:1) is added to each sample weh, and the fluorescence polarization is measured using a LJL Analyst (LJL Biosystems, Sunnyvale, CA).

Denaturing Gradient Gel Electrophoresis

Denaturing gradient gel electrophoresis (DGGE) is a gel system which ahows electrophoretic separation of DNA fragments differing in sequence by a single base pair. The separation is based upon differences in the temperature of strand dissociation of the wild-type and mutant molecules. During electrophoresis, fragments migrating through the gel are exposed to an increasing concentration of denatarant in the gel. When the DNA fragments are exposed to a critical level of denaturant, the DNA strands begin to dissociate. This dissociation causes a significant reduction in the mobihty of the fragment. The position in the gel at which the level of denatarant is critical for a particular DNA fragment is a function of the Tm of the DNA fragment and is therefore different for wild-type versus mutant fragments. Consequently, upon migration to the position at which the level of denaturant is at the critical point, for either the wild-type or the mutant fragment, the mobihty of these two molecules wih become different, thus resulting in their separation. The mutation detection rate of DGGE approaches 100%. Although the technique of DGGE is relatively simple to perform, and does not require radioisotopes or toxic chemicals, it does require some speciahzed equipment. Furthermore, DGGE can only be used to analyze fragments between 100 and 800bp due to the resolution limit of polyacrylamide gels. DGGE is advantageous over other methods useful for detecting sequence variations because the behavior of DNA molecules on DGGE gels can be modeled by computer thereby making it possible to accurately predict the detectabihty of a mutation in a given fragment. Genomic DNA fragments can be efficiently transferred from the gel fohowing DGGE as described in US Patent No. 5,190,856.

Chemical Cleavage of Mismatches

Chemical cleavage of mismatch (CCM) is another technique for detection of sequence variations that is useful according to the invention. CCM is based upon the abihty of hydroxylamine and osmium tetroxide to react with the mismatch in a DNA heteroduplex and the abihty of piperidine to cleave the heteroduplex at the point of mismatch. According to the method of CCM, sequence variations are detected by the appearance of fragments that are smaher than the untreated heteroduplex fohowing denaturing polyacrylamide gel electrophoresis. DNA fragments up to lkb in size can be analyzed by CCM with a probable 100% detection rate for sequence variation. CCM is particularly useful for either detecting ah of the sequence variations in a particular fragment of DNA or for determining that there are no sequence variations in a particular fragment of DNA.

Constant Denaturant Capillary Electrophoresis (CDCE) Analysis

CDCE analysis is particularly useful in high throughput screening, i.e., wherein large numbers of DNA samples are analyzed. CDCE analysis combines several elements of both replaceable linear polyacrylamide capillary electrophoresis and constant denatarant gel electrophoresis. The technique of CDCE is a rapid, high resolution procedure that demonstrates a high dynamic range, and is automatable. The method of CDCE, as described in detail in Khrapko et al, 1994, Nucleic Acids Res. 22:364, involves the use of a zone of constant temperature and a denaturant concentration in capillary electrophoresis. Linear polyacrylamide gel electrophoresis is performed at viscosity levels that permit facile replacement of the matrix after each run. For a typical 100 bp fragment of DNA, point mutation-containing heteroduplexes are separated from wild type homoduplexes in less than 30 minutes. Using laser- induced fluorescence to detect fluorescent-tagged DNA, the system has an absolute limit of detection of 3 x 10⁴ molecules with a linear dynamic range of six orders of magnitude. The relative limit of detection is about 3/10,000, i.e., 100,000 mutant sequences are recognized among 3 x 10⁸ wild type sequences. This approach is applicable to analysis of low frequency mutations, and to genetic screening of pooled samples for detection of rare variants.

Rnase Cleavage

An additional method for genotyping that is useful according to the invention is RNase Cleavage. Various ribonuclease enzymes, including RNase A, RNase TI and RNase T2 specifically digest single stranded RNA. When RNA is annealed to form double stranded RNA or an RNA/DNA duplex, it can no longer be digested with tliese enzymes. However, when a mismatch is present in the double stranded molecule, cleavage at the point of mismatch may occur.

RNase Cleavage is preferably performed with RNase A. Ribonuclease A specifically digests single stranded RNA but can also cleave heteroduplex molecules at the point of mismatch. The extent of cleavage at single base mismatches depends on both the type of mismatch, and the sequence of DNA flanking the mismatch. Sequence variations leading to mismatch are indicated by the presence of fragments that are smaher than the uncleaved heteroduplex on denaturing polyacrylamide gels. According to the invention, RNase Cleavage involves forming a heteroduplex between a radiolabeled single stranded RNA probe (riboprobe) and a PCR product derived from a biological sample. If a point mutation is present in the PCR product, fohowing treatment of the resulting RNA/DNA heteroduplex with RNase A, the RNA strand of the duplex maybe cleaved. The sample is then denatured by heating and analyzed on a denaturing polyacrylamide gel. If the RNA probe has not been cleaved, it wih be the same size as the PCR product. If the probe has been cleaved, it wih be smaher than the PCR product. RNase Cleavage can be used to easily detect a 1 bp deletion.

However, smah insertions may not be as easily detected as smah deletions, by RNASE Cleavage, as 'looping-out' occurs on the target strand rather than the probe strand.

Heteroduplex Analysis Another method for genotyping according to the invention is heteroduplex analysis.

Heteroduplex molecules, i.e., double stranded DNA molecules containing a mismatch, can be separated from homoduplex molecules on ordinary gels. The exact rate of detection of sequence variations by heteroduplex analysis is unknown, but is clearly significantly lower than 100%.

Presumably, the sequence of DNA flanking the mismatch, rather than the actual mismatch affects the detectabihty. Mismatches that are located in the middle of a DNA fragment are detected most easily.

Although heteroduplex analysis is less sensitive than some of the other genotyping methods described, it maybe considered useful according to the invention due to its simplicity. Mismatch Repair Detection (MRD)

Another technique that is useful for genotyping according to the invention is mismatch repair detection (MRD). MRD is an in vivo method that detects DNA sequence variation by the occunence of a change in bacterial colony color. DNA fragments to be screened for variation are cloned into two MRD plasmids, and bacteria are transformed with heteroduplexes of these constructs. The resulting colonies are blue in the absence of a mismatch and white in the presence of a mismatch. MRD can be used to detect a single mismatch in a DNA fragment as large as 10 kb in size. MRD permits high- throughput screening of genetic mutations, and is described in detail in Faham et al, 1995, Genome Research 5:474.

Mismatch Recognition by DNA Repair Enzymes

Another technique that is useful for detecting sequence variations according to the invention is Mismatch Recognition by DNA Repair Enzymes. The E.coh mismatch correction systems are well- understood. Three of the proteins required for the methyl-directed DNA repair pathway: MutS, MutL and MutH are sufficient to recognize 7 of the possible 8 single base-pah mismatches (C/C mismatches are not recognized) and cut/nick the DNA at the nearest GATC sequence. The MutY protein, which is involved in a distinct repair system can also be used to detect A/G and A/C mismatches. Some mammalian enzymes are also useful for mismatch recognition: thymidine glycosylase can recognize ah types of T mismatch and 'all-type endonuclease' or Topoisomerase I is capable of detecting ah 8 mismatches, but does so with varying efficiencies, depending on both the type of mismatch and the neighboring sequence.

The MutS gene product is the methyl-directed repair protein which binds to the mismatch. Purified MutS protein has been used to detect mutations by several different methods. Gel mobihty assays can be performed in which DNA bound to the MutS protein migrates more slowly through an acrylamide gel than free DNA. This method has been used to detect single base mismatches.

An alternative method for the use of MutS in mismatch recognition, which does not require gel electrophoresis, involves the immobihzation of MutS protein on nitrocehulose membranes. Labeled heteroduplexed DNA is used to probe the membrane in a dot-blot format. When both DNA strands are used, ah mismatches can be recognized by binding of the DNA to the protein attached to the membrane. Although C/C mismatches are not detected, the corresponding G/G mismatch derived from the other strand is recognized. This technique is particularly useful because it is simple, inexpensive, and amenable to automation. However, the detection efficiency of this method maybe limited by the size of the DNA fragment. In particular, this method works weh for very short fragments.

Sequencing by Hybridization (SBH)

An alternative method for detecting sequence variations according to the invention is sequencing by hybridization (SBH). According to this method, arrays of short (8-10 base long) ohgonucleotides are immobilized on a sohd support in a manner similar to the reverse dot-blot protocol, and probed with a target DNA fragment. In particular, ohgonucleotides are synthesized together and directly onto the support.

The synthesis system begins with a sihcon chip coated with a nucleotide hnked to a light- sensitive chemical group which is used to ihuminate particular grid co-ordinates removing the blocking group at these positions. The chip is then exposed to the next photoprotected nucleotide, which polymerizes onto the exposed nucleotides.

In this manner, as a result of successive rounds of nucleotide additions, ohgonucleotides of different sequences can be synthesized at different positions on the sohd support. Thirty-two cycles of specific additions (i.e., 8 additions of each of the four nucleotides) should enable the production of ah 65,536 possible 8-mer ohgonucleotides at defined positions on the chip.

When the chip is probed with a DNA molecule, e.g., a fluorescently labeled PCR product, fully matched hybrids should give a high intensity of fluorescence and hybrids with one or more mismatches should give substantially less intense fluorescence. The combination of the position and intensity of the signals on the chip enables computers to derive the sequence of the DNA molecule being analyzed for the presence of sequence variations.

Ahele-Specific Ohgonucleotide Hybridization

The technique of ahele-specific ohgpnucleotide (ASO) hybridization or the 'dot-blot' is also useful for genotyping according to the invention. Under specific hybridization conditions, an ohgonucleotide wih only bind to a PCR product if the two are 100% identical. A single base pah mismatch is sufficient to prevent hybridization. A pah of ohgonucleotides, one carrying the wild type base and the other caπying a single base change, as compared to the wild type sequence, can be used to determine if a PCR product is homozygous wild type, heterozygous or homozygous mutant for a particular base change. When performing conventional dot blots, the PCR product is fixed onto a nylon membrane and probed with a labeled ohgonucleotide. When performing a 'reverse dot blot' , an ohgonucleotide is fixed to a membrane and probed with a labeled PCR product. The probe may be isotopicahy labeled, or non-isotopicahy labeled. The technique allows for the genotyping of multiple PCR amplified samples for the presence of a single base change.

Allele-Specific PCR

Many methods for identifying sequence variations involve the analysis of PCR-amphfied DNA. The ahele-specific polymerase chain reaction (also cahed the amphfication refractory mutation system or ARMS) comprises an assay that occurs during the PCR reaction itself. ARMS requires the use of sequence-specific PCR primers which differ from each other at their terminal 3 ' nucleotide and are designed to amplify only the normal ahele in one reaction, and only the mutant ahele in another reaction. When the 3' end of a specific primer is 100% identical to the target, amphfication occurs. When the 3' end of a specific primer is not 100% identical to the target, amphfication does not occur. Agarose gel electrophoresis is used to detect the presence of an amplified product. The genotype of a (heterozygous) wild-type sample is characterized by amphfication products in both reactions, and a homozygous mutant sample generates product in only the mutant reaction.

This technique can be modified so that the 5' ends of the ahele-specific primers are labeled with different fluorescent labels, and the 5' end of the common primers are biotin labeled. According to this alternate protocol, the wild-type specific and the mutant-specific reactions are performed in. a single tube. The advantages of this approach are that a gel electrophoresis step is not required, and the method is amenable to automation.

Primer-Introduced Restriction Analysis

The method of primer-introduced restriction analysis (PIRA) can also be used for genotyping according to the invention. PIRA is a technique which ahows known sequence variations to be detected by restriction digestion. By introducing a base change close to the position of a known sequence variation (for example by using a PCR primer containing a mismatch, as compared to the target sequence), it is possible to create a restriction endonuclease recognition site that indicates the presence of a particular sequence change. The combination of the altered base in the primer sequence and the altered base at the mutation site, creates a new restriction enzyme target site. This approach maybe used to create a new restriction enzyme site in either the wild-type ahele or the mutant ahele. If a novel restriction enzyme site is introduced in the mutant ahele then, fohowing digestion with the appropriate restriction enzyme, the homozygous wild-type form would produce a single band of the fuh-length size, the homozygous mutant form would produce a single band of the reduced size and the heterozygous form would produce both full length and reduced sized bands. Band size wih be analyzed by gel electrophoresis. Ohgonucleotide Ligation Assay

The technique of ohgonucleotide hgation can also be used for genotyping according to the invention.

The method of ohgonucleotide hgation is based on the following observations. If two ohgonucleotides are annealed to a strand of DNA and are exactly juxtaposed, they can be joined by the enzyme DNA hgase. If there is a single base pair mismatch at the junction of the two ohgonucleotides then Hgation wih not occur. According to the method of ohgonucleotide hgation, the two ohgonucleotides used in the assay are modified by the addition of two different labels. According to this method, the assay for a hgated product involves detecting a hgated product by assaying for the appearance of the labels of the two ohgonucleotides on a single molecule rather than visuahzation of a new, larger sized DNA fragment by gel electrophoresis.

When hgation reactions are conducted in 96- weh microtiter plates and hgation is scored by ELISA, the ohgonucleotide hgation assay can be performed by a robot and the results can be analyzed by a plate reader and fed directly into a computer. This method is therefore extremely useful for detecting the presence of a sequence variation in a large number of samples. The ohgonucleotide hgation assay is performed on PCR-amphfied DNA. A modification of this assay, termed the hgase chain reaction, is performed on genomic DNA and involves amphfication with a thermostable DNA . hgase.

Direct DNA Sequencing

Genotyping according to the invention may also be carried out by directly sequencing the DNA sample in the region of the gene of interest, using DNA sequencing procedures weh-known in the art (described above in Section D, entitled "Isolation of a Wild Type Gene").

Mini-Seqnencing

The technique of mini-sequencing (also known as single nucleotide primer extension) can also be used to detect any known point mutation, deletion or insertion, according to the invention. Obtaining sequence information for just a single base pah only requires the sequencing of that particular base. This can be done by including only one base in the sequencing reaction rather than ah four. When this base is labeled and complementary to the first base immediately 3 ' to the primer (on the target strand), the label wih not be incorporated. Thus, a given base pah can be sequenced on the basis of label incorporation or failure of incorporation without the need for electrophoretic size separation. 5' Nuclease Assay

Genotyping according to the invention can also be performed by the method of 5' nuclease assay. The 5' nuclease assay is a technique that monitors the extent of amphfication in a PCR reaction on the basis of the degree of fluorescence in the reaction mix. A low level of fluorescence indicates no amphfication or very poor amphfication and a high level of fluorescence indicates good amphfication. This system can be adapted to permit identification of known sequence variations, without the need for any post-PCR analysis other than fluorescence emission analysis.

PCR amphfication is detected by measuring the 5' to 3 ' exonuclease activity of Taq polymerase. Taq polymerase cleaves 5' terminal nucleotides of double stranded DNA. The prefeπed substrate for Taq polymerase is a partiahy double stranded molecule. Taq polymerase cleaves the strand that contains the closest free 5' end. According to the 5' nuclease assay, an ohgonucleotide 'probe' which is phosphorylated at its 3' end so as to render it incapable of serving as a DNA synthesis primer, is included in the PCR reaction. The probe is designed to anneal to a position between the two amphfication primers. When an actively extending Taq polymerase molecule reaches the probe molecule, it partiahy displaces the probe and then cleaves the probe at or near the single stranded/double stranded cleavage site until the entire probe is broken up and removed from the template. The polymerase continues this process of displacement and cleavage until the entire probe is broken up and removed from the template. The probe is labeled in a manner that permits detection of the removal of the probe. In particular, the probe is labeled at different positions with two different fluorescent labels. One label has a localized quenching effect on the fluorescence of the other

(reporter) label. This effect is mediated by energy transfer from one dye to the other, and requires that the two dyes are in close proximity to each other. If the probe is cleaved at a position between the reporter and the quencher dyes, the two dyes become physically separated thereby resulting in an increase in fluorescence which is proportional to the yield of the PCR product.

Representational Difference Analysis (RDA)

Genotyping according to the invention can also be caπied out by Representational Difference Analysis (RDA). RDA is described in detail in Lisitsyn et al, 1993, Science 259:946, and an adaptation which combines selective breeding with RDA is described in Lisitsyn et al., 1993, Nature Genet. 6:57. RDA identifies sequence dissimilarities through the apphcation of a powerful approach to subtractive hybridization. According to the method of RDA, one first creates simplified representations, cahed amplicons, from two samples that are being compared. An amplicon can comprise, for example, the set of BglH fragments that are smah enough to be amplified by the PCR. The iterative subtraction step begins with the hgation of a special adaptor to the 5' end of fragments contained in the amphcon derived from the test sample (tester amphcon). The tester amphcon is then melted and briefly reannealed in the presence of a large excess of amphcon, derived from the wild type sample (driver amphcon). Those tester fragments that reanneal (presumably fragments absent from the wild type, driver amphcon) can serve as a template for the addition of the adaptor sequence to the 3 '-end of the "partner" fragment. As a result, these tester fragments can be exponentiahy amphfied by PCR. This procedure is then repeated to achieve successively higher enrichment.

RDA may be used to clone sequences that are either whohy absent from the wild type sample or are present in the wild type DNA, but are contained in a restriction fragment that is too large to be amphfied in the amphcon. The former case may arise from a total deletion; the latter from a restriction fragment length polymorphism with the short ahele present in the tester but not the wild type DNA. RDA is useful for subtracting DNA from an individual with a particular disease from normal DNA so as to identify regions showing homozygous or heterozygous deletions; locating fragments present in a parent with a dominant disorder but absent in his unaffected offspring; and locating mRNAs expressed in normal tissue but not present in tissue isolated from an individual with a particular disease.

Denaturing High Performance Liquid Chromatography

According to the scanning method of Denaturing High Performance Liquid Chromatography (DHPLC), partial heat denaturation and a linear acetonitrile column are used to identify polymorphisms in DNA fragments. DHPLC provides a method of comparative DNA sequencing based on the capability of ion-pah reverse phase hquid chromatography on alkylated nonporous poly(styrene divinylbenzene) particles to resolve homo- from heteroduplex molecules under conditions of partial denaturation. This method can potentiahy be automated to ahow for rapid analysis of a large - number of samples (Underhih et al, 1996, Proc. Natl. Acad. Sci. USA, 93:196).

Mass Spectroscopy

Matrix-assisted laser desorption-ionization-time-of-fhght (MALDI-TOF) mass spectroscopy is another method according to the invention by which genotyping can be performed. The method of MALDI-TOF mass spectroscopy is based on the irradiation of crystals formed by suitable smah organic molecules (refened to as the matrix) with a short laser pulse at a wavelenght close to the resonant adsorption band of the matrix molecules. This causes an energy transfer and desorption process producing matrix ions. Low concentrations of nucleic acid molecules are added to the matrix molecules while in solution and become embedded in the sohd matrix crystals upon drying of the mixture. The intact nucleic acids are then desorbed into the gas phase and ionized upon irradiation with a laser allowing their mass analysis. MALDI is used primarily with time-of-flight spectrometers where the time of flight is related to the mass-to-charge ratio of the nucleic acids molecules. Reviewed in Griffin TJ. and Smith L.M., 2000, Trends Biotech 18:77. Genotyping can be performed by any of the fohowing MALDI-TOF mass spectroscopy approaches including sequencing of PCR products (Fu, D-J et al, 1998, Nat. Biotechnol. 16:381; Kirpekar, F. et al, Nucleic Acids Res. 26:2554), direct mass-analysis of PCR products (Ross, P.L. et al, 1998, Anal. Chem. 70:2067), analysis of ahele-specific PCR (Taranenko, N.I. et al, 1996, Genet. Anal. Biomol Eng. 13:87) or LCR (hgase chain reaction; Jurinke, C. et al, 1996, Anal. Biochem. 237:174) products, analysis of RFLP-PCR products (Srinivasan, J.R. et al, 1998, Rapid Commun. Mass Spectrom. 12:1045), minisequencing (Haff, L.A. and Smirnov, IP., 1997, Genome Res. 7:378; Higgens, G.S. et al, 1997, BioTechniques 23:710), analysis of PNA (peptide nucleic acid) hybridization probes (Griffin, TJ. et al, 1997, Nat. Biotech. 15:1368; Ross, P.L., Anal. Chem. 69:4197; Jiang- Baucom, P. et al, 1997, Anal. Chem. 69:4894), or direct analysis of invasive cleavage products (Griffin, TJ. et al, 1999, Proc. Natl Acad. Sci. USA 96:6301).

6. Methods of Specifying a Polymorphism

The invention provides methods for specifying a particular polymorphism. By "specifying an polymorphism" is meant defining a polymorphism in the context of a larger region of nucleic acid ^' which contains the polymorphism, and is of sufficient length to be easily differentiated from any other position in the genome.

A unique nucleotide position (e.g. a polymorphic site) in the human genome can be specified by describing a unique sequence of DNA within the genome, and providing the location of the unique nucleotide position relative to that sequence. Preferably this is done by providing the sequence identity of a length of unique DNA containing the polymorphism, and indicating which of the nucleotide sites is polymorphic.

A calculation can be made to determine a sequence length which wih be unique in the 3 billion nucleotide human genome. If it is assumed that the genome contains equal numbers of the nucleotides A, G, C and T, and that they occur randomly in the genome, one can determine the probabihty of any given sequence of a defined length occurring in the genome; a random 12mer wih appear in a random 3,000,000,000 bp genome 179 times, a random 15 mer wih appear in a random 3,000,000,000 bp genome 3 times and a random lόmer wih appear in a random 3,000,000,000 bp genome 1 time.

Thus, it would appear that specifying 16 bp would uniquely define a sequence in the genome. However, the genome is not composed of random sequence and does not contain equal amounts of A, G, C and T. In fact, 10-12 bp sequences are likely to be specific for 95% of genes. Some sequences may even be specified by as few as 8 nucleotides. The minimum sequence length that is useful according to the invention for identifying polymorphisms in most gene and intergenic sequences is approximately 9-15 bp.

In the case of repeat sequences and sequences associated with gene famihes, the probabihty of observing a particular sequence is greatly increased and it becomes difficult to specify a polymorphism in the context of a sequence that is only on the order of 9-15 bp. There are many types of repeats including tandem repeats, where a larger sequence block has within it smaher repeat units (e.g. microsatehites). Tandem repeats usuahy occur within non-genic areas, but can also occur within genes and subsequently affect gene function; they can be 10-lOOOs of bp long, or, if located in centromeres and telomeres, be megabase sized. Some repeats are composed of blocks which do not have sub-repeat units and are non-functional (e.g. -300 bp Alu repeats). These occur by duphcation/dispersal throughout the genome. It may be difficult to specify a polymorphism that occurs in a gene that is a member of a gene family. Through the mechanism of gene duphcation, gene famihes, comprising multiple copies of a gene in which some, but not ah of the DNA sequence has diverged, have been formed. Thus, certain regions of a gene may be conserved in different gene family members: With time, a duphcated gene can lose function and the sequence of the duphcated gene can deteriorate; the amount of homology between the original gene and the duphcated version depends upon the time since duphcation. Other duplications maintain function and retain some level of similarity with the original gene in the important domains. Some related genes can share nearly 100% homology across a region that is hundreds of bp long, and yet have no significant homology at any other location. In these cases, it may be necessary to specify dozens or more nucleotides to provide a unique sequence. To identify a unique sequence, a search must be done wherein a specific sequence is compared to ah known human sequences and the minimum unique sequence is defined. However, in the absence of a complete sequence for the human genome, it cannot be guaranteed that a sequence is truly unique. Empirical experimentation can be used to determine the minimum sequence for specificity/uniqueness. In the case of a gene family member, if sequence information is available for the region conesponding to the region of interest in other members of the gene family, than it may be possible to define a unique short (9-15 bp) sequence that contains a polymorphism and has specificity. In the event that a particular region cannot be defined as unique, a larger region of nucleic acid which contains the polymorphism wih be required to define a polymorphism in a gene that is a member of a gene family. It is predicted that a sequence of 9-15 bp wih be sufficient to define a polymorphism in 99% of all cases.

Methods of specifying a polymorphism that involve using sequences which either encompass or overlap the polymorphic site to be tested or do not encompass or overlap the polymorphic site to be tested are useful according to the invention and are described below.

Ohgonucleotide Hybridization.

An ohgonucleotide is designed such that it is specific for a target sequence, and hybridizes only at the target sequence site. This ohgonucleotide wih not hybridize if the target sequence differs at the position in the sequence to be tested. Another ohgonucleotide is designed such that it hybridizes with the polymorphic form of the sequence. A DNA sample is tested for hybridization with each of the two probes independently. If the DNA hybridizes to only one of the probes, it can be concluded that the individual is homozygous for the conesponding sequence. If both probes hybridize to a test DNA sample, then the individual is heterozygous. Hybridization wih be detected by the method of Southern blot analysis (as described in Section C entitled "Production of a Nucleic Acid Probe").

Specifying a Polymorphism by PCR

An alternative method for specifying a particular polymorphism involves a PCR-based strategy. According to this method, a region of a candidate gene to be tested is amphfied by PCR (as described). The amphfied fragment is digested with a restriction enzyme that wih not cut a fragment that contains a polymorphism, due to the location of the polymorphism wittiin the recognition site of this restriction enzyme. The products of the digestion reaction mixture are size separated in an agarose gel, stained with ethidium bromide, and visualized under ultraviolet hght to determine if the amphfied product has been digested. According to this method, the PCR primers provide the specificity for a particular polymorphism by virtue of the specific sequence of the two primers, as weh as by the location of the primer binding sites in the target DNA. Although, multiple sites for primer binding may exist in a target DNA sequence, only the sites that are close enough together wih produce an amphfied product that includes the nucleic acid region containing the polymorphism.

Alternatively, a PCR reaction is caπied out with PCR primers that contain polymorphisms. According to this embodiment, if the template nucleic acid lacks the polymorphism present in the primers there wih be no PCR product. Thus, according to this embodiment of the invention, the absence of a PCR product indicates that a polymorphism is not present in the target sequence. Primer Extension

A DNA fragment comprising the region containing a polymorphism is PCR amphfied from an individual to be tested. The PCR product is denatured and one strand is retained for analysis. An ohgonucleotide probe is designed such that it is specific for a region in the sequence and hybridizes such that its 3' terminal nucleotide is paired with the nucleotide adjacent to the one to be tested. The PCR product and probe are combined with a polymerase and terminating, differentially colored, nucleotides. The polymerase extends the probe by one base, and only the base which is complementary to the site being tested is added. The reaction is washed, and the color of the reaction indicates the nucleotide that has been added and the sequence at the position of interest. The PCR step provides one level of specificity by amplifying a region (1 - 10000 bp as deshed between the PCR primers) from a complex (3,000,000,000 bp) mixture. The PCR probes primers must be unique in both their hybridization specificity and their proximity to one another. Since proximity of the two PCR primers is needed (i.e. a distance across which a polymerase can extend to join the primers), shorter PCR primers can be used, e.g. in theory a smah enough region could be amphfied with a 8-10 bp binding site for a PCR primer. To ensure that a primer hybridizes with specificity, a primer must be at least 5 bp.

A second level of specificity is provided by the primer which is extended in the primer extension reaction. Since this primer is hybridizing to a short piece of DNA, it can be short and unique for the fragment with which it binds. The primer is at least 5bp and preferably 8bp. Although the primer used for the primer extension step is located probe adjacent to the polymorphic site, the PCR primers should not overlap with the polymorphic site being tested.

Southern Blotting

One method for detecting a previously defined polymorphism involves Southern blot analysis of wild type and mutant DNA fohowing digestion with a restriction enzyme which has a recognition sequence which includes the polymorphic site to be tested. According to this method, a particular restriction enzyme cuts wild type DNA but does not cut mutant DNA due to the presence of a polymorphism within the recognition site of this restriction enzyme. Many restriction enzymes exist which recognize 4bps. The resulting fragments wih be size separated in an agarose gel, transferred to a membrane and probed with a nucleic acid probe. If the site is uncut, the fragment is one length and if the site is cut the fragment wih be of a shorter length.

The nucleic acid hybridization probe wih provide specificity to the particular polymorphism being tested by defining the polymorphism in the context of a larger stretch of nucleic acid sequence. The nucleic acid probe may comprise the nucleic acid sequence corresponding to the region known to contain the polymorphism. The sequence-specific probe may be located 10, 100, 1000, or even 100s of thousands of bases from the region containing the polymorphism. If the probe is located some distance from the region containing the polymorphism, an intervening recognition site for the restriction enzyme cannot be located between the probe hybridization site and the region of interest containing the polymorphism site. Typically, a hybridization probe useful according to this method wih be much larger than the minimum length of a sequence (9-15 bp) required to give specificity to, or define a particular polymorphism.

Alternatively, a chemical or enzyme which recognizes a unique pah of nucleotides at the site of a polymorphism, can be used to detect the polymorphism. According to this method, the amount of sequence required for recognition by a chemical or enzyme is 2 bp (providing that the 2 bp sequence is unique in a region large enough to produce a fragment which can then be bound by a specific probe).

According to a variation of the above method, a labeled chemical or enzyme which binds to one sequence of the polymorphic recognition site and not another is used. This method involves the steps of digesting the DNA with a restriction enzyme, and adding a labeled, sequence-specific binding protein (e.g. a restriction enzyme that lacks cleavage capability). The sequence-specific binding protein wih bind to multiple sites in the genome, including the site to be tested. The fragments wih be separated on a gel and then probed with a probe specific for the test sequence. If the fragment identified by the second probe is identical to a fragment identified by the first probe (e.g. the labeled chemical or enzyme), then the sequence being tested for is present.

7. Determination of the Phenotypic Outcome of a Polymorphism

To determine the phenotypic outcome of a polymorphism according to the invention, it is necessary to screen suitable populations to obtain a statisticahy significant measure of the association of a polymorphism with a particular disease (e.g osteoarthritis). The invention provides methods for performing polymorphism genotyping in appropriate populations (described above). The invention also provides in vitro and in vivo assays useful for determining the phenotypic outcome of a polymorphism in a candidate gene. Every polymorphism has the potential to alter the genetic activity of an individual. At the level of a single gene, the effect of a polymorphism can range from an inconsequential, silent change to a change that causes a complete loss of protein function to a gain of abenant or detrimental function mutation. The severity of the effect of a polymorphism on gene activity wih depend on the exact molecular consequences of the particular polymorphism. For example, alterations of a single pre- mRNA sphcing dinucleotide could have profound effects on both the quantitative and qualitative properties of gene activity since alterations in sphcing efficiency can both reduce the overall level of normal transcription as weh as cause "exon skipping". If the deleted exon involves a coding exon then exon skipping wih lead to an alteration in the amino acid composition of the resulting protein and likely effect protein activity. To accurately asses the role of a particular polymorphism in the regulation of various molecular events, appropriate assays for both gene expression and protein function must be carried out.

In vitro assays useful for determining the effects of a polymorphism on gene expression and protein function include, but are not limited to the fohowing. i. Transcriptional Regulation

The transcriptional regulation of a candidate gene containing a polymorphism may be altered, as compared to the wild type gene.

Promoter Activity

If a polymorphism is located in the promoter, enhancer or repressor region of a candidate gene, promoter assays (weh known in the art) wherein the altered promoter of the candidate gene is used to drive the expression of a reporter gene (e.g. CAT, luciferase, GFP) are performed. Changes in the transcriptional regulation of a candidate gene due to the presence of a polymorphism can also be detected by methods useful for measuring the level of mRNA including S 1 nuclease mapping and RT- PCR.

SI Analysis

The SI enzyme is a single-stranded endonuclease that wih digest both single-stranded RNA and DNA. According to the method of S 1 analysis, a probe that has been efficiently labeled to a high specific activity at the 5' end through the use of a kinase, is used to determine either the amount of an mRNA species or the 5' end of a message. A single stranded probe that is complementary to the sequence of the RNA species of interest is utihzed in S 1 analysis. If the structure of a particular mRNA species is known, SI analysis is performed with ohgonucleotide probes of at least 40 bp, that are complementary to the RNA of interest. It is preferable to use ohgonucleotides wherein the 5' end of the ohgonucleotide is complementary to the RNA. It is also preferable to use ohgonucleotides wherein the 5' terminal residues contain dG or dC residues. If Si nuclease analysis wih be utihzed to determine the 5' termii of an RNA species, the 3' end of the ohgonucleotide should extend at least 4 nucleotides beyond the RNA coding sequence. The inclusion of additional nucleotides facihtates differentiation of a band resulting from an R A:DNA duplex and a band representing the probe.

A hybridization probe for SI analysis is prepared by incubating 2ρmol of an ohgonucleotide in the presence of 150 mCi[y³²P]ATP (3000-7000Ci/mmol), 2.5 ml 10X T4 polynucleotide kinase buffer (700mM Tris-Cl, pH 7.5, 100 mM MgCl₂, 50 mM ditMothreitol, 1 mM spermidine-Cl, 1 mM EDTA), and 10U T4 polynucleotide kinase for 37°C for 30-60 minutes. The radiolabeled probe is ethanol precipitated and resuspended at lml/0.3ng ohgonucleotide or 10⁵ cpm.

The hybridization reaction is performed as fohows. An amount of probe equal to 5x10⁴

Cerenkov counts is added to 5Omg RNA on ice and ethanol precipitated. The resulting pehet is resuspended in 20ml SI hybridization solution (80% deionized formamide, 40 mM PIPES, pH 6.4,

400mM NaCl, 1 mM EDTA, pH 8), denatured for 10 min at 65°C and hybridized overnight at 30°C.

The fohowing day, 300 ml of a mixture of 150 ml 2x SI nuclease buffer (0.56M NaCl, 0.1 M sodium acetate, pH 4.5, 9mM ZnSO₄), 3ml 2mg/ml single-stranded cah thymus DNA, 147 ml H,0 and 300U

SI nuclease is added to the hybridization reaction and incubated for 60 minutes at 30°C. Fohowing the addition of 80ml SI stop buffer (4M ammonium acetate, 20mM EDTA, 40 mg/ml tRNA) the sample is ethanol precipitated, resuspended in formamide loading dye, denatured and analyzed on a denaturing polyacrylamide/urea gel of the appropriate percentage for the expected size of the protected band

(Ausubel et al, supra).

RT-PCR

The method of RT-PCR is useful according to the invention for RNA expression analysis. According to the method of reverse transcription /polymerase chain reaction (RT-PCR) during the reverse transcription (RT) step, the RNA is converted to first strand cDNA, which is relatively stable and is a suitable template for a PCR reaction. In the second step, the cDNA template of interest is amphfied using PCR. This is accomplished by repeated rounds of annealing sequence- specific primers to either strand of the template and synthesizing new strands of complementary DNA from them using a thermostable DNA polymerase.

An RNA sample is ethanol precipitated with a cDNA primer. It may be preferable to use a cDNA primer that is identical to one of the amphfication primers. To the pehet is added 12 ml H-0, 4ml 400mM TrisCl, pH 8.3 , and 4 ml 400 mM KCl The mixtare is heated to 90°C, slow cooled to 67°C, microfuged and incubated for 3 hours at 52°C. Fohowing the addition of 29ml reverse transcriptase buffer (per sample/2.5ml 400mM TrisCl, pH8.3, 2.5ml 400mM KCl, 1ml 300mM MgCl₂, 5ml lOOmM DTT, 5ml 5mM 4 dNTP mix, 2ml actinomycin D, 11ml H-0) and 0.5ml (16U) AMV reverse transcriptase, the sample is incubated for 1 hour at a temperatare between 37°C and 55°C. The temperatare wih be adjusted in accordance with the composition of the primer and the RNA of interest. The sample is then extracted sequentiahy with phenol and chloroform, and ethanol precipitated. The resulting cDNA pehet is resuspended in 40ml ELO. 5ml of the cDNA sample is mixed with 5ml or each amphfication primer (~20mM each), 4ml 5mM 4dNTP mix, 10ml 1OX amphfication buffer (500mM KCl, lOOmM TrisCi, pH8.4, lmg/ml gelatin) and 70.5ml ILO. After the mixture is heated for 2 minutes at 94°C, 0.5 ml (2.5U) Taq DNA polymerase is added and the sample is overlaid with mineral oil PCR amphfication of the cDNA wih be performed using the fohowing automated amphfication cycles: 39 cycles (2 minutes at 55°C, 2 minutes at 72°C, 1 minute at 94°C), 1 cycle (2 minutes at 55°C, 7 minutes at 72°C). The number of cycles can be varied in accordance with the abundance of RNA (Ausubel et al., supra).

If a polymorphism is located in a transcription factor binding site, assays including but not limited to the yeast two-hybrid assay (Fields et al, 1994, Trends Genet., 10:286) can be used to determine the effects of a polymorphism on transcription factor binding. If the protein product of the gene of interest is a DNA binding protein the phenotypic outcome of a polymorphism maybe npahed nuclear transport, DNA binding, chromatin assembly or chromatin structure, methylation or histone deacetylation.

Nuclear Transport hi iunocytochemical methods or ceh fractionation techniques (as described above) are used to determine if the protein is correctly locahzed in the nucleus.

The DNA binding properties of a transcription factor are determined by gel shift analysis (as described in Ausubel et al, supra), ohgonucleotide selection, southwestern assays or by hnmunohistochemical analysis of fixed chromosomes.

Gel Shift Analysis

The method of gel shift analysis is used to detect sequence specific DNA-binding proteins from crude extracts. According to this method, proteins that bind to an end-labeled DNA fragment wih retard the mobihty of the fragment. The change in the mobihty of the labeled fragment is detected by the appearance of a discrete band comprising the DNA-protein complex.

A number of methods for preparing nuclear and cytoplasmic extracts useful for gel shift analysis are known in the art. For example, nuclear extracts are prepared according to the fohowing method. A ceh pehet is washed in PBS, resupended in a volume of hypotonic buffer (10 mM HEPES, pH 7.9, 1.5 mM MgCl₂, lOmM KCl, 0.2 mM PMSF, 0.5 mM DTT ) that is approximately equal to 3 times the packed ceh volume and ahowed to swell on ice for 10 minutes. Cehs are homogenized in a glass Dounce homogenizer and the nuclei are cohected by centrifugation and resupended in a volume of low-salt buffer (20 mM HEPES, pH 7.9, 25% (v/v) glycerol, 1.5 mM MgCl₂, 0.02 M KCl, 0.2 mM EDTA, 0.2 mM PMSF, 0.5 mM DTT) equivalent to one-hah of the packed nuclear volume. Fohowing the addition of a volume of high-salt buffer (20 mM HEPES, pH 7.9, 25% (v/v) glycerol, 1.5 mM MgC-₂, 1.2 M KCl, 0.2 mM EDTA, 0.2 mM PMSF, 0.5 mM DTT) equivalent to one-hah of the packed nuclear volume (dropwise with stiπing) to the nuclei, nuclear extraction is carried out for 30 minutes with continuous gentle stiπing. The nuclei are cohected by centrifugation and the nuclear extract is dialyzed against 50 volumes of dialysis buffer (20 mM HEPES, pH 7.9, 20% (v/v) glycerol, lOOmM KCl, 0.2 mM EDTA, 0.2 mM PMSF, 0.5 mM DTT) until the conductivities of extract and buffer are equivalent. The extract is removed from the dialysis tubing and analyzed for protein concentration (Ausubel et al, supra).

Probes useful for gel shift analysis include a fragment of plasmid DNA or a gel-purified double stranded ohgonucleotide. Preferably the probe is labeled with Klenow fragment by incubating a 100ml solution of plasmid DNA or ohgonucleotide with lOOmCi of the deshed [a-³²P] dNTP, 4ml of 5 mM 3 dNTP mix and 2.5 U Klenow fragment for 20 minutes at room temperatare. Upon the addition ; of 4ml of a solution comprising 5 mM of the dNTP corresponding to the radioactive dNTP, the sample is incubated for 5 minutes at room temperature. The radiolabeled probe is ethanol precipitated, resuspended in TE buffer and gel purified.

Gel shift analysis is performed by incubating 10,000 cpm of the labeled probe (0.1-0.5 ng) with 2mg poly (dl-dC)-poly(dl-dC), 300 mg BSA, and approximately 15mg of a nuclear extract or buffered crude protein extract prepared, for example, as described above, for 15 minutes at 30°C. An ahquot of the binding reaction is analyzed by electrophoresis on a prewarmed low-ionic strength gel (e.g. a 4% polyacrylamide gel in TBE) and autoradiography (Ausubel et al, supra).

Oligoselection Assays for DNA Binding Activity

DNA binding activity is an essential property of proteins involved in many basic ceh biological events, such as chromatin structure, transcriptional regulation, DNA replication and repair. The biological activity of a DNA binding protein can be assayed by defining the optimal target DNA binding site. Using the PCR based primer selection technique (Blackwell, 1990, Science, 250:1104) the canonical nucleotide sequence defining the binding site is elucidated in vitro by mixing purified full length protein, or just the DNA binding domain of a protein of interest, with an ohgonucleotide duplex pool containing a completely randomized central region flanked by primer-annealing sites. Multiple rounds of immunoprecipitation and amphfication by PCR enriches for high affinity sites which are cloned are sequenced in order to define a canonical binding site.

The abihty of a DNA binding protein to correctly regulate chiOmathi assembly and structure can be determined by DNase hypersensitivity assays. Alternatively, coimmunoprecipitation experiments or Western blot analysis can be used to determine if the DNA binding protein is associated with a component of the chromatin.

Southwestern Blot Assay for Protein-DNA Interactions The abihty of a protein to bind DNA is measured by using the "Southwestern" blot technique

(for example see Antahs et al, 1993, Gene, 134:201). According to this method, radiolabehed DNA is incubated with protein that has been immobilized on nitrocellulose filters and the amount of bound DNA is measured by scintihation counting or autoradiography fohowed by densitometry. The protein to be tested can be pure protein, immunoprecipitated protein, crude ceh lysates or even recombinant protein denatured directly from bacterial colonies, yeast or ceh culture.

Assay of Protein Binding to Chromosomes in Vivo: Immunocytology of Fixed Chromosomes

Numerous biologically important nuclear proteins are in direct contact with genomic DNA. The presence of these proteins can be detected immunocytologicahy by fixing metaphase chromosomes such that the protein is permanently fixed at the region of DNA to which it normahy binds. The presence and cytological location of the protein can then be determined by incubating the fixed chromosomes with an antibody directed against the protein of interest, and performing standard methods of immunohistochemical staining (Zink and Paro, 1989, Nature, 337:468).

Coimmunoprecipitation Assay for Chromatin Assemhly/Structure

If an antibody specific for a protein of interest exists, immunoprecipitation can be used to test for the presence of the protein (Otto and Lee, 1993, Methods CehBiol, 37:119, Banting, 1995, In Gene Probes 1: A practical approach. Chapter 8: Antibody probes, pp. 225-227, IRL press.). The fohowing methods are used for deteπriining if a protein of interest is associated with a particular subcehular component. According to one method, proteins are immunoprecipitated with an antibody specific for a cellular component (e.g. chromatin or nuclear antigens), the immunoprecipitated material is analyzed on a gel by denaturing polyacrylamide gel electrophoresis and western blot analysis is performed with an antibody specific for the protein of interest, to determine if a physical association exists between the cehular component and the protein of interest. Various incubation and wash treatments of the ceh lysate are used to remove background contamination and enhance the sensitivity of detection (Banting, 1995, supra). Alternatively, the initial immunoprecipitation can be carried out with the antibody specific for the protein of interest, and the western blot analysis can be performed with an antibody specific for a cehular component. According to a variation of this method, prior to immunoprecipitation the cehs can be treated with a protein crosslinker to ensure that protein-protein interactions are maintained during immunoprecipitation. According to another variation of this method, proteins can be cross-linked to DNA and then precipitated (Dedon et al, 1991, Anal. Biochem., 197:83). If DNA coprecipitates with a particular protein, this suggests that DNA is associated with, and presumably bound to the protein. The coprecipitating DNA can be sequenced to identify the bound sequence.

DNAse Hypersensitivity

The transcriptionahy active promoter region of a gene can be analyzed for susceptibihty to cleavage by DNAsel (Montecino et al , 1994,Biochemistry, 33 :348). Efficient cleavage of genomic DNA is dependent on the accessibility of this enzyme to the DNA, and is influenced by several factors, including nucleosome packaging, overah chromatin configuration, and the presence of DNA binding proteins such as transcription factors. DNA sequence variations within the promoter DNA may have profound effects on these factors and result in aberrant regulation of gene transcription and ultimately abnormal biological activity of the gene. Therefore, altered gene activity around a polymorphic site can be detected as increased or decreased DNAsel hypersensitivity (Vaishnaw et al, 1995, Immunogenetics, 41:354).

Assay for DNA Methylation Accurate mapping of DNA methylation patterns, for example, in CpG islands which are unmethylated regions of DNA, is used to investigate and gain a better understanding of diverse biological processes such as the regulation of imprinted genes, X chromosome inactivation and tumor suppressor gene silencing inhuman cancer. DNA methylation at specific sites is most frequently studied by use of methylation-sensitive restriction endonucleases (for example Hpalf) and Southern blotting (Sambrook et al, supra). The sensitivity of this method can be enhanced several hundred-fold by performing a hgation-mediated PCR step (as described in Steigerwald et al, 1990, Nucleic Acids Res., 6:1435) after enzyme treatment. An alternative strategy termed methylation-specific PCR (Herman et al, 1996, Proc Natl Acad Sci USA., 93:9821), is used to determine the methylation status of CpG islands without the use of methylation-specific restriction enzymes.

Histone-Deacetylation

Transcription of chromatin-packaged genes involves highly regulated changes in nucleosome structure that control DNA accessibihty. Changes in nucleosome structure can be mediated by enzymatic complexes which control the acetylation and deacetylation of histones. Transcription elongation is required for the formation of the unfolded structure of transcribing nucleosomes, and histone acetylation is required for the maintenance of these structures (Walia et al, 1998, J. Biol. Chem., 3:14516). Deacetylation can be prevented by incubating cehs with histone deacetylase inhibitors such as sodium butyrate or trichostain A. To assay for changes in acetylation and the state of transcriptional activity, chromatin fractions are purified using organomercury and hydroxylapatite dissociation chromatographic techniques (Waha et al, supra).

ii. Transcription Start Site

To determine if a particular polymorphism causes a change in the transcriptional start site of a candidate gene S 1. nuclease mapping and primer extension can be performed. The presence of a polymorphism may cause an mRNA to be aberrantly expressed. In particular, a polymorphism may change the tissue specificity or developmental expression pattern of an mRNA species. A variety of molecular methods for detecting mRNA known in the art can be performed to determine the expression pattern of an mRNA These methods include, but are not limited to the fohowing: Northern blot analysis, RT-PCR, SI analysis, RNase Protection analysis, or in situ hybridization analysis of sections, wherein the samples are derived from multiple different tissues or from a tissue at different stages of development. Northern blot analysis, RT-PCR and S 1 analysis can also be used to determine if a polymorphism results in an altered pattern of mRNA sphcing.

Northern-B 1 otting

The method of Northern blotting is weh known in the art. This technique involves the transfer of RNA from an electrophoresis gel to a membrane support to ahow the detection of specific sequences in RNA preparations.

Northern blot analysis is performed according to the fohowing method. An RNA sample (prepared by the addition of MOPS buffer, formaldehyde and formamide) is separated on an agarose/formaldehyde gel in IX MOPS buffer. Fohowing staining with ethidium bromide and visuahzation under ultra violet hght to determine the integrity of the RNA, the RNA is hydrolyzed by treatment with 0.05M NaOH/1.5MNaCl fohowed by incubation with 0.5M Tris-Cl (pH 7.4)/1.5M NaCl. The RNA is transferred to a commerciahy available nylon or nitrocellulose membrane (e.g. Hybond-N membrane, Amersham, Arlington Heights, IL) by methods weh known in the art (Ausubel et al, supra, Sambrook et al, supra). Fohowing transfer and UV cross linking, the membrane is hybridized with a radiolabeled probe in hybridization solution (e.g. in 50% formamide/2.5% Denhardt's/100-200mg denatured salmon sperm DNA/0. 1% SDS/5X SSPE) at 42°C. The hybridization conditions can be varied as necessary as described in Ausubel et al, supra and Sambrook et al, supra. Fohowing hybridization, the membrane is washed at room temperatare in 2X SSC/0.1% SDS, at 42°C in IX SSC/0.1% SDS, at 65°C in 0.2X SSC/0.1% SDS, and exposed to film. The stringency of the wash buffers can also be varied depending on the amount of background signal (Ausubel et al, supra).

RNase Protection Analysis RNase Protection analysis can be used to analyze RNA structure and amount and determine the endpoint of a specific RNA.

The method of RNase protection is more sensitive than SI analysis since it utihzes a sequence . specific hybridization probe that is labeled to a high specific activity. The probe is hybridized to sample RNAs and treated with ribonuclease to remove free probe. Fohowing ribonuclease treatment, the fragments comprising probe annealed to homologous sequences in the sample RNA are recovered by ethanol precipitation, and analyzed by electrophoresis on a sequencing gel. The presence of the target mRNA is indicated by the presence of an appropriately sized fragment of the probe.

A probe is labeled by the method of in vitro transcription (in the presence of [a-³²P] CTP as described in Section B entitled "Production of a Polynucleotide Sequence". The RNA sample to be analyzed is ethanol precipitated and resuspended in 30ml hybridization buffer (4 parts formamide/1 part 200 mM PIPES, pH 6.4, 2 M NaCl, 5 mM EDTA) containing 5 x 10⁵ cpm of the probe RNA. The mixtare is denatured 5 minutes at 85°C and incubated at the deshed hybridization temperature (30°C to 60°C) for >8 hours. To each reaction mixture is added 350 ml ribonuclease digestion buffer (10 mM Tris-Cl, pH 7.5, 300 mM NaCl, 5 mM EDTA) containing 40 mg/ml ribonuclease A and 2 mg/ml ribonuclease TI. The sample is incubated for 30-60 minutes at 30°C. Fohowing the addition of 10 ml 20%SDS and 2.5ml 20 mg/ml proteinase K, the sample is incubated for 15 minutes at 37°C. The sample is extracted with phenol /cMoroformhsoamyl alcohol, ethanol precipitated, resuspended in RNA loading buffer (80% (v/v) formamide, 1 mM EDTA, pH 8.0, 0.1 % bromophenolblue, 0.1 % xylene cyanol), denatured and analyzed by electrophoresis on a denaturing polyacrylamide/urea gel and autoradiography (Ausubel et al, supra).

Primer Extension The method of primer extension is used to map the 5 ' end of an RNA and to quantitate the amount of an RNA of interest by using reverse transcriptase to extend a primer that is complementary to a region of a given RNA.

An ohgonucleotide primer is labeled in a kinase reaction as described for SI analysis. The primer extension reaction is performed by mixing 10-50 mg total cehular RNA (in lOml) with 1.5ml 10X Hybridization buffer (1.5M KCl, 0. IM TrisCl, pH 8.3 , lOmM EDTA) and 3.5 ml labeled ohgonucleotide. Samples are heated to 65°C for 90 minutes and ahowed to slow cool at room temperatare. To each sample is added 30 ml of primer extension reaction mixtare (0.9 ml Tris-Cl, pH 8.3, 0.9 ml 0.5M MgCl₂, 0.25 ml DTT, 6.75 ml 1 mg/ml actinomycin D, 1.33 ml 5 mM 4dNTP mix, 20 ml H-0, 0.2ml 25 U/ml AMV reverse transcriptase). Samples are incubated for 1 hour at 42°C, and then, fohowing the addition of 105 ml RNase reaction mix (100 mg/ml salmon sperm DNA, 20 mg/ml RNase A) for 15 noάnutes at 37°C. Samples are extracted in phenol/chloroformhsoamyl alcohol, ethanol precipitated, resuspended in stop/loading dye (20 mM EDTA, pH 8.0, 0.05% bromophenol blue, 0.05% xylene cyanol in formamide), heated at 65°C and analyzed by electrophoresis on a 9% acrylamide/7M urea gel and autoradiography.

In Situ Hybridization

Cytological techniques weh known in the art can be used to determine the temporal and spatial expression patterns of mRNA (in situ hybridization of tissue sections) and protein (immunohistochemistry in individual cehs).

Preparation of histological samples

Tissue samples intended for use in in situ detection of either RNA or protein are fixed using conventional reagents; such samples may comprise whole or squashed cehs, or sectioned tissue. Fixatives useful for such procedures include, but are not hmited to, formalin, 4% paraformaldehyde in an isotonic buffer, formaldehyde (each of which confers a measure of RNAase resistance to the nucleic acid molecules of the sample) or a multi-component fixative, such as FAAG (85 % ethanol, 4% formaldehyde, 5% acetic acid, 1% EM grade glutaraldehyde). For the detection of RNA, water used in the preparation of an aqueous component of a solution to which the tissue is exposed until it is embedded is RNAase-free, i.e. treated with 0.1% diethylprocarbonate (DEPC) at room temperatare overnight and subsequently autoclaved for 1.5 to 2 hours. Tissue wih be fixed at 4°C, either on a sample roller or a rocking platform, for 12 to 48 hours in order to ahow the fixative to reach the center of the sample. Prior to embedding, excess fixative wih be removed and the sample wih be dehydrated by a series of two- to ten-minute washes in increasingly high concentrations of ethanol, beginning at 60% and ending with two washes in 95% and another two in 100% ethanol, fohowed by two ten-minute washes in xylene. Samples wih be embedded in one of a variety of sectioning supports, e.g. paraffin, plastic polymers or a mixed paraffin/polymer medium (e.g. Paraplast®Plus Tissue Embedding Medium, supphed by Oxford Labware). For example, fixed, dehydrated tissue wih be transfened from the second xylene wash to paraffin or a paraffin/polymer resin in the hquid-phase at about 58°C. The paraffin or a paraffin/polymer resin wih be replaced three to six times over a period of approximately three hours to dilute out residual xylene. The sample wih be incubated overnight at 58°C under a vacuum, in order to optimize infiltration of the embedding medium into the tissue. The next day, fohowing several additional changes of medium at 20 minute to one hour intervals, also at 58°C, the tissue sample wih be positioned in a sectioning mold, the mold wih be surrounded by ice water and the medium wih be ahowed to harden. Sections of 6mm thickness wih be taken and affixed to 'subbed' shdes, which are slides coated with a proteinaceous substrate material, usuahy bovine serum dbumin (BSA), to promote adhesion. Other methods of fixation and embedding are also apphcable for use according to the methods of the invention; examples of these are found in Humason, G.L., 1979, Animal Tissue Techniques, 4th ed. (W.H. Freeman & Co., San Fransisco), as is frozen sectioning (Senano et al, 1989, supra).

In situ Hybridization Analysis According to the method of in situ hybridization a specifically labeled nucleic acid probe is hybridized to cehular RNA present in individual cehs or tissue sections. In situ hybridization can be performed on either paraffin or frozen sections. Depending on the deshed sensitivity and resolution, either film or emulsion autoradioagraphy can be utihzed to detect the hybridized radioactive probe.

The fohowing method of in situ hybridization is performed by incubating shdes containing ceh or tissue specimens in a shde rack contained within a glass staining dish. According to this method, it is preferable to use solutions that have been prepared fresh. Prior to the hybridization steps, shdes are dewaxed to remove the sectioning support material. The dewaxing protocol involves sequential washes in xylene, rehydration by sequential washes in 100%, 95%, 70% and 50% ethanol, and denaturation in 0.2N HCl. Fohowing a heat denaturation step (70°C in 2X SSC), samples are postfixed in a freshly prepared solution of 4% PFA, washed in PBS, incubated in 10 mM DTT (10 min at 45°C) and blocked in 400 ml PBS containing 0.617g DTT, 0.74 g iodoacetamide and O.Sg N-ethylmaleimide, for 30 min at 45°C in a water bath covered with aluminum foil, due to the hght sensitivity of iodoacetamide and N-ethylmaleimide. The samples are washed in PBS and equihbrated sequentiahy in freshly prepared 0. IM trieflianolamine (TEA buffer), TEA buffer/0.25% acetic anhydride, and TEA buffer/0.5% acetic anhydride. Fohowing a blocking step in 2X SSC, the sample are dehydrated by sequential washes in 50%, 70%, 95%, and 100% ethanol and ah dried. ³⁵S-labeled riboprobes and competitor probes prepared in the absence of a radiolabel (prepared as described in Section B entitled "Production of a Polynucleotide Sequence") or double-stranded DNA probes (prepared with

[³⁵S]dNTPs by methods weh known in the art including nick translation or random oligonucleotide- primed synthesis) are heated to 100°C for 3 min and diluted to a concentration of 0.3 mg/ml final probe concentration, in 50% formamide, 0.3M NaCl, lOmM TrisCl, pH 8.0, 1 mM EDTA, lx Denhardt solution, 500 mg/ml yeast tRNA, 500 mg/ml ρoly(A) (Pharmacia), 50 mM DTT, 10% polyethylene glycol (MW 6000). The hybridization step is carried out by covering the sample with an appropriate amount of probe, and incubating for 30 min to 4 hour at 45°C in a chamber designed to prevent dilution or concentration of the hybridization solution. Samples are washed sequentiahy at 55°C in solution A , . (50% (v/v) formamide, 2X SSC, 20 mM 2-mercaptoethanol), and solution B (50% (v/v) formamide, . 2X SSC, 20 mM 2-mercaptoethanol 0.5% (v/v) Triton-X-100) and at room temperatare in solution C (2X SSC, 20 mM 2- mercaptoethanol). Fohowing a 15 minute incubation with RNase, samples are washed at 50"C in solution C, and at room temperature in 2X SSC. Samples are rehydrated by sequential washes in 50% ethanol/0.3M ammonium acetate, 70% ethanol/0.3M ammonium acetate, 95% ethanol/0.3M ammonium acetate, and 100% ethanol. Shdes are ah dried and analyzed by film or by emulsion autoradiography (Ausubel et al, supra).

hi. mRNA Stabihty/Control of Turnover and mRNA Transcription Rate

Changes in mRNA stability/control of turnover and mRNA transcription rates due to the presence of a polymorphism, can be detected by the fohowing methods.

mRNA Stabihty

Gene-expression can be regulated by variations in mRNA stabihty (Liebhaber, 1997, Nucleic Acids Symp Ser., 36:29 and Ross J. 1996, Trends Genet, 5:171). Any gene variation occurring within the cis-acting elements which control mRNA abundance may influence gene expression levels (Peltz et al, 1992, Curr Opin Ceh Biol, 4:979). Quantitative RT-PCR (Kohler, et al, 1995, Quantitation of mRNA by polymerase chain reaction, Springer) and mRNA radiolabelling techniques are two methods for measuring relative mRNA abundance and stabihty. Quantitative PCR employs an internal standard to provide a direct comparison between alternative reactions, enabling comparison of low abundance transcripts or transcripts derived from a sample that is only available in a limited quantity (McPherson MJ et al, eds, 1995, PCR2- A practical approach. IRL Press).

Assay for mRNA Transcription Rates Genetic polymorphism within the regulatory regions of a gene can significantly alter transcription rate and mRNA stabihty, resulting in reduced biological activity of the encoded protein. One of the most sensitive assays for measuring the rate of gene transcription is the nuclear runoff assay (Groudine and Casimir, 1984, Nucleic Acids Res 12: 1427). Nuclei isolated from ceh lines expressing the target gene of interest are treated with radiolabehed UTP and the level of incorporation of radiolabel into nascent RNA transcripts is determined by filter hybridization to immobihzed cDNA derived from the target gene.

iv. Intracellular mRNA Localization

A genetic variation can cause a change in the locahzation of a particular mRNA species (e.g. to the cytoskeleton, or to the nuclear scaffold).

ImmunoMstochenύsitry

Changes in RNA locahzation can be detected by immunohistochemical methods weh known in the art (e.g. in situ analysis described above).

Oocyte Injection Assays

In many cases mRNA, like protein, is localized in relation to the polarity of the ceh or the cytoskeletal architecture (St. Johnston, 1995, Ceh, 81:161). T &Xenopus oocyte is a popular, experimentally tractable, system for studying intracehular trafficking of mRNA (Nakielny et al, 1997, Annu. Rev. Neurosci, 20:269). Fluorescently labehed RNA is microinjected into the large oocyte ceh where its location can be detected using standard microscopy methods. Polymorphic variants of a particular mRNA species may differ in their response to cehular mechanisms responsible for partitioning mRNA within the ceh. This method has been useful for demonstrating that sequence variations can affect sub-cellular locahzation (Grimm et al, 1997,EMBO J., 16:793)

v. Post-Translational Alterations

Post-Translational alterations resulting from premature stop codons, translational readthrough or multiple open reading frames and translational suppression may occur as a result of a polymorphism. To detect post-translational alterations, a polynucleotide comprising one or more polymorphisms is subjected to in vitro transcription and in vitro translation (as described in sections B and J entitled "Production of a Polynucleotide Sequence" and "Preparation of a Labeled Protein").

The translation product(s) are analyzed for the appearance of aberrantly sized proteins. Additional post-translational alterations that may occur as a result of a polymorphism include changes in locahzation due to an altered signal sequence, and changes in glycosylation, myristilation, and susceptibihty to or sites of proteolytic cleavage.

The method of immunocytochemistry can be used to determine if a protein is incoπectly localized, due to the presence of an altered signal sequence.

Immunohistochemistry l niunonistochemical techniques including indirect immunofluorescence, immunoperoxidase labeling or immunogold labeling, are used for protein locahzation.

Immunofluorescent labeling of tissue sections (prepared as for in situ analysis, described above) is performed by the fohowing method. Shdes containing the sample of interest are equihbrated to room temperatare washed in PBS, incubated with an appropriate dilution of primary antibody (1 hour at room temperature), washed in PBS, incubated with an appropriate dilution of secondary antibody (1 hour at room temperatare), washed in PBS and analyzed under a microscope (Ausubel et al. , supra). Alternatively, the sensitivity of the immunohistochemical reaction is increased by using a streptavidin-secondary antibody conjugate reacted with a biotinfluorochrome conjugate. Alternatively, immunogold labeling is used to detect a protein of interest by using an immunogold-conjugated secondary antibody.

Immunoperoxidase labeling of tissue sections is performed by the fohowing method. Shdes are pretreated in 0.25% hydrogen peroxide, incubated with primary antibody, washed in PBS and incubated (1 hour at room temperature) with a specific secondary bridging antibody capable of recognizing both the primary antibody and a Horseradish peroxidase antiperoixidase (PAP) complex.

The shdes are washed in PBS and developed in diaminobenzidene substrate solution (0.03% (w/v) 3,3' diaminobenzidene in 200 ml PBS) at room temperature (Ausubel et al, supra). Alternatively, protein locahzation is determined by ceh fractionation wherein cehs are biosyntheticahy labeled, the labeled material is fractionated, and the radiolabeled proteins in each fraction are analyzed by immunoprecitation with an antibody specific for the protein of interest.

Assay for Glycosylation Mhibition

Changes in protein glycosylation can be detected by radiolabelhng a protein of interest with sugars, determining if a change in the cehular locahzation (by immunocytochemistry) of the protein in culture has occurred due to aberrant glycosylation, or by determining the effects of inhibitors of glycosylation on the migration pattern of proteins analyzed by polyacrylamide gel electrophoresis. Post-translational glycosylation of proteins plays an important role in defining protein function

(Baeziger, 1994, FASEB J., 13:1019; Jacob, 1995, Curr. Opin. Struct. Biol, 5:605). Protein glycosylation can be inhibited by tanicamycin, an antibiotic, as weh as by several sugar analogues (Schwarz, 1991, Behring Inst Mitt., 89:198). These reagents are used to characterize the effects of sequence changes on protein glycosylation.

Assay for Post-Translational Modification with Lipids

Changes in protein modification with hpids (e.g. myristilation) are detected by radiolabelhng a - protein of interest with myristic acid or by determining if a change in the cehular locahzation of the protein in culture has occuπed as a result of aberrant hpid modification (by immunocytochemistry). Covalent attachment of hpids is a mechanism by which eukaryotic cehs direct and, in some cases, control, membrane locahzation of proteins (Casey, 1994, Cun. Opin. Ceh. Biol, 2:219). Such post-translational addition of myristyl, palmityl or prenyl side-chains has a key role in the functional regulation of many proteins (Chow et al, 1992, Curr. Opin. Ceh. Biol, 4:629; Resh, 1994, Ceh, 763:411). Assays for detecting proteins that are covalently modified by the attachment of hpids include labeling with [³H]myristate (Stevenson et al, 1992, J. Exp. Med., 176:1053), or a combination of enzymatic and chemical cleavage techniques performed in conjunction with tandem mass spectrometry to determine sites of modification (Papac et al, 1992, J. Biol. Chem., 267:16889).

Proteolytic Cleavage Post-translational cleavage of polypeptides is an important mechanism for modulating protein function in many physiological processes. Protease activity is involved in zymogen processing, activation of enzyme catalysis, tissue/ceh remodeling, signal transduction cascades, protein degradation and ceh death pathways (Rappay, 1989, Prog Histochem Cytochem., 18:1). A protein that is predicted to be a protease or the target of a protease can be assayed in vitro using purified proteins or ceh extracts (Muta et al, 1995, J. Biol. Chem. 270:892) where cleavage efficiency is monitored by standard PAGE or western blotting. Alternatively, proteases and/or their targets can be expressed from expression plasmids in in vivo ceh culture systems in order to monitor their biological activity (Zhang, et al, 1998, J. Biol. Chem. 273:1144). The specificity of proteolytic cleavage is determined using irjhibitors that selectively block seine, cysteine, aspartic and metaho proteolytic activity (e.g. pepstatin A selectively inlhbits aspartic proteases) (Rich, et al, 1985, Biochemistry., 24: 3165).

To determine if a protein has been modified such that the sites of proteolytic cleavage have been altered, or susceptibihty to proteolytic cleavage has changed pulse chase experiments with radiolabeled protein can be carried out to determine the precursor-product relationship fohowing digestion with a protease of a given specificity. The method of pulse chase labeling is described in Ausubel et al, supra. Alternatively, inhibitors of proteases (e.g acid proteases or seine proteases) can be used to identify protease cleavage sites.

vi. Changes in Receptor Properties

If the gene of interest encodes a receptor protein, a polymorphism may modify the properties of the receptor such that receptor binding/turnover or activation is altered. Receptor formation can be hnpahed if a polymorphism causes improper receptor locahzation or assembly.

Receptor Locahzation

To determine if a receptor protein is being expressed at the proper location (e.g. nucleus, cytoplasm, ceh surface), the receptor can be localized by immunocytochemical techniques. Alternatively, cehs that are expressing the receptor can be fractionated and subjected to Western blot analysis or biosyntheticahy labeled, fractionated and analyzed by immunoprecipitation.

Protein-Protein Interactions/In vitro Assembly Assays for Receptors

A number of methods can be used to determine if a receptor is colocahzed with the appropriate protein partner.

The function of a protein may be dependent on the abihty of the protein to interact with other proteins as part of a large complex. For example, certain ceh surface receptors consist of a receptor complex that is composed of several homo- or heteromeric protein subunits, and activation by hgand can result in altered protein-protein interactions both within the receptor complex and with "downstream" targets such as G-proteins (Okada and Pessin, 1996, J. Biol. Chem., 271:25533). Protein-protein interactions can be assayed immunologically by coimmunoprecipitation of native (Gilboa et al, 1998, J. Biol. Chem., 140:767) or chemically cross-linked complexes (Haniu et al, 1997, J. Biol. Chem., 272:25296), or through protein-protein mobihty shift assays (Stern and Frieden, 1993, Anal. Biochem., 212:221). If ah of the components of a receptor complex have been identified, one can employ in vitro reconstitation assays to assess whether a single protein alteration can effect the functioning of the entire complex (Durovic et al, 1994, J. Biol. Chem., 269:30320).

Assay for In Vitro Assembly of Multimeric Protein Complexes

To determine whether these genetic variations have affected protein complex assembly, experiments are carried out wherein recombinant mutant subunits are transfected into cehs and coexpressed with the other subunit components in vitro. Proper assembly is assessed by immunoprecipitation of the protein complex in question with antibodies specific for the various members of the complex fohowed by PAGE analysis (Koster et al, 1998, Biophysl J., 74:1821).

Assay Receptor Binding/Turnover

Receptor-hgand interaction is essential for the functionality of the bound complex. Genetic changes that alter either hgand or receptor can dramatically affect receptor binding, turnover, and subsequent activation of downstream signaling events. Receptor binding/turnover can be measured by standard Scatchard analysis of radiolabehed hgand binding in vitro (Culouscou et al, 1993, J. Biol Chem. 268:10458) or in cehular based assays (Greenlund et al, 1993, J. Biol. Chem. 268: 18103).

Ligand Binding as Measured by Affinity Chromatography

Alternatively, affinity chromatography methods (weh known in the art) can be employed to determine if a receptor is demonstrating abeπant binding characteristics. According to the method of affinity chromatography, receptor-hgand interactions are ahowed to occur, and the binding efficiency or receptor and hgand and/or turnover of receptor-hgand complexes is measured. Alternatively, affinity chromatography can be used to isolate one or more components of a receptor hgand interaction for further analysis (March et al, 1974, Adv. Exp. Med. Biol, 42:3). The method of affinity chromatography typically involves immobilizing on a sohd support one component, for example a known hgand for a receptor, and then incubating the immobihzed hgand with radiolabehed protein under optimal binding conditions. To measure the exact binding affinity of a given ligand-receptor pah, an increasing amount of non-labeled competitor is added. This assay can be used to assess altered binding efficiency resulting from the presence of a polymorphism in a protein of interest. Receptor Activation Assays: Phosphorylation, Kinase Activity and Mitogenic Stimulation

Almost ah signaling that occurs through ceh surface receptors is regulated by phosphorylation, a reversible post-translational event that occurs at specific amino acid residues and is catalyzed by a protein kinase activity present within the receptor itself (autophosphorylation) or in trans via direct interaction with an associated kinase (Hunter, 1997, Philos Trans R Soc Lond B Biol Sci., 353:583). The specific effect of phosphorylation on a biological activity depends on the receptor, but often results in modulation of endogenous receptor kinase activity or interaction with associated proteins, which are also often kinases. The results of a phosphorylation event are passed on through a cascade of protein kinases/phosphatases which ultimately effect downstream processes controlling gene transcription, ceh prohferation, metabohsm, movement and differentiation (Patarca, 1996, Crit Rev Oncog., 7:343). The biological function of a receptor is usuahy assayed in ceh culture fohowing over- expression. The phosphorylated state of a receptor can be assayed directly by immunological methods by employing an antibody that specifically recognizes a phosphorylated residue (Bangalore, 1992., Proc Natl Acad Sci USA., 89:11637). Endogenous kinase activity associated with a receptor is measured via the incorporation of radiolabehed phosphate in immunoprecipitated receptor complex (Kazlauskas and Cooper, 1989, Ceh 58:1121). "Downstream" events of receptor activity including mitogenic stimulation or map kinase activity, can be measured by tritiated thymidine incorporation (Luo et al, 1996, Cancer Res. 56:4983), or by mobihty-shift analysis of map kinase on western blots (Vietor, 1993., J. Biol Chem. 268:18994), respectively. Ixnmunocytochemical methods can be used to determine if a receptor-hgand complex is conectly translocated to the nucleus. Alternatively, nuclear preparations (prepared as described below) can be analyzed by Western blot or immunoprecipitation for the presence of the receptor protein.

If a receptor is a transcriptional activator, the abihty of the receptor to induce gene expression can be measured by a variety of methods including Northern blot analysis, or reporter gene assays wherein the promoter region isolated from a gene that is activated by the receptor regulates the expression of a reporter protein.

vii. Enzyme Catalysis The gene of interest may encode a protein that has an enzymatic activity wherein the enzyme catalyzes a reaction that is critical to the general metabohsm of a ceh. To determine if a mutated protein is hnpahed in its enzymatic function, assays can be performed to measure the enzymatic activity of the protein. There are many important enzymatic activities associated with normal cehular metabohsm, including: glycosidation, esterification, amidation, hydroxylation, acetylation, sulfonylation, alkylation. Each of these activities are assayed using in vitro methods employing overexpressed or purified proteins, weh known in the art (Eisenthal and Danson, 1992, Enzyme Assays: A Practical Approach, Rickwood et al, Eds., IRL Press. Oxford, England). The protein of interest may also be involved in various aspects of DNA synthesis or replication. In vitro assays for the enzymatic reactions involved in DNA synthesis or replication (e.g. polymerase, hgase, exonuclease or helicase activity) are known in the art. The biological activity of the proteins catalyzing these activities are assayed in vitro using standard enzymatic techniques (Adams, 199, DNA Replication: A Practical Approach I, Rickwood, et al, Eds., IRL Press. Oxford, England). If the protein of interest is involved in glycolysis or energy transport, assays for measuring transporter activity or the activity of ATP dependent pumps are useful, according to the invention, for determining if a mutated protein is npahed in these functions.

Transporter Activity Mammalian cehs possess a variety of transporter systems, for example amino acid transporters, which have overlapping substrate specificity (Van Winkle et al, 1993, Biochim Biophys , Acta, 1154: 157). To determine if a polymorphism in a candidate gene of interest has altered the function of the protein product of this gene as a molecular transporter, the tall-length cDNA clone is isolated by standard expression cloning strategies, and a change in activity of the fuh-length cRNA or antisense cRNA upon microinjection into Xenopus laevis oocytes is determined by measuring changes in influx/efflux transport of radiolabehed amino acid molecules (Broer et al, 1995, Biochem J., 312(Pt 3):863), neurotransmitters or their metabohtes.

ATP-dependent pumps Activity Mammalian cehs possess a variety of molecules that are categorized as ATP-binding cassette or ATP-dependent transporters or pumps. These include the Na⁺-K⁺-ATPase ion pump, the calcium uptake pump, (K⁺ + H⁺)-ATPase and the human multidrug resistant protein termed P-glycoprotein. Alterations in pump activity are investigated by expressing the clone specific for the pump protein(s) of interest in Xenopus oocytes, and performing tracer studies which measure the changes in ATP- dependent uptake or extrusion of a radiolabehed substrate, and changes in the coupling ratios (e.g. moles substrate transported/mole ATP hydrolyzed) (Shapiro et al., 1998, Eur. J. Biochem., 254:189).

viii. Ion Channel The gene of interest may encode for a protein that is a component of an ion channel. Immunocytochemical methods can be used to determine if an ion channel protein demonstrates the appropriate ceh type specificity.

The activity of an ion channel can be measured by electrophysiological methods in oocytes. Alternatively, the sensitivity of ion channel activity to a particular inhibitor can be determined.

Assays for Ion Channel Activity in Oocytes

Polymorphisms which alter ion channel function and regulation are studied using the oocytes of Xenopus laevis. Injection of the oocytes with exogenous in vitro transcribed mRNA results in the production and functional expression of foreign membrane proteins, including voltage- and neurotransmitter- operated ion channels (Dascal et al, 1987., CRC Crit Rev Biochem., 224:317). Changes in the oocyte transmembrane current in response to expression of an exogenous mRNA is measured. This technique has been improved by the development of rapid superfusion systems that utihze a dual role perfusion micropipette that controls internal solution as weh as monitoring voltage (Costa et al, 1994, Biophys J., 67:395). This technology represents a useful system for studying various aspects of ion channels encoded for by foreign rnRNAs including channel expression, single- channel behavior, and the response of channels to the action of pharmacologically active substances (Sigel, 1987 J. Physiol, 386: 73).

Patch Clamp Assays for Ion Channel Activity

The function of individual channel proteins is determined by the high resolution patch clamp technique. This technique (which is useful in a variety of ceh types, including Xenopus oocytes described above) involves measuring changes in transmembrane cunent across the ceh membrane in vitro (Sachs et al, 1983, Methods Enzymol., 103: 147). Processes such as signaling, secretion, and synaptic transmission are examined at the cehular level by the patch clamp method. The gene expression pattern and protein structure of ionic channels can be deteπnined by combining information derived from high-resolution electrophysiological recordings obtained by the patch clamp method with molecular biological analysis (Liem et al, 1995, Neurosurgery, 36: 382).

A polymorphic variation in a gene that encodes a protein that is a member of a multimeric protein complex, such as an ion channel or a cytoskeletal structural component, can alter the assembly and function the multimeric protein complex (Lee et al, 1994., Biophys J., 66: 667). A gene variation may affect protein-protein interaction, or disrupt the production of components of a multimeric complex, thereby disrupting stoichiometry and consequently decreasing stabihty. Assay for In Vitro Assembly of Multimeric Protein Complexes

In vitro assembly assays (described above) can be performed to determine if a polymorphism has affected the assembly of an ion channel.

ix. Cehular Properties

The influence of a polymorphism on general aspects of ceh behavior, including ceh morphology, adhesive properties, differentiation and prohferation can be assessed using a combination of methods including microscopic observation of ceh cultures (Azuma et al, 1994, HistolHistopathol, 9:781), immunohistochemistry, and FACs analysis techniques (Beesley, 1993, hmminocytochemistry: a Practical Approach, Rickwood, et al, (Eds), IRL Press and Ormerod, 1994, Flow Cytometry: a practical Approach, Rickwood et al, (Eds), IRL Press. Oxford, England).

Assays for Measuring Apoptosis

Apoptosis has been implicated in the etiology and pathophysiology of a variety of human diseases. Gene variants which influence the process of apoptosis can be assessed by a variety of methods of analysis involving either the tissues or cehs (Allen et al, 1997, J Pharmacol Toxicol Methods, 37: 215). Ceh cultares expressing the gene variants of interest are analyzed using Annexin V ^': which interacts strongly with phosphatidylserine residues that have been exposed as a result of plasma membrane breakdown occurring in the early stages of apoptosis. Either vital or fixed material can be analyzed by Annexin V labeling in combination with microscopy and flow cytometry detection methods (van Engeland et al, 1998, Cytometry, 31:1). TdT-mediated deoxyuridine triphosphate (dUTP)-biotin nick end-labeling (TUNEL) is a prefened method for specific staining of apoptotic cehs i histological sections and cytology specimen (Labat-Moleur et al, 1998, J. Histochem Cytochem., 46:327; Sasano et al, 1998., Diagn Cytopathol, 18:398). Apoptosis is also detected by quantification of DNA fragmentation by ethidium bromide staining and gel electrophoresis, or by the use of sataration labeling of 3' ends of DNA fragments (Peng and Liu, 1997, Lab Invest., 77:547).

Assay for In Vivo Receptor Function: Growth Cone Guidance Assay

Activation of ceh-surface receptors can result in the stimulation of ceh motihty. There are many different famihes of signaling molecules, for example the netrins, (Serafini et al., 1994, Ceh. 78: 409), which are responsible for both contact mediated or chemo-mediated attraction and repulsion of rnigrating cehs. A classic model for this activity is the trajectory that the leading edge "growth cone" takes when a neuron is stimulated to grow out from explanted neural tissue in ceh culture (Goodman, 1996, Annu Rev Neurosci. 19: 341). Ligands present in the culture medium or immobihzed on a substrate bind to receptors on the ceh-surface of the growth cone and trigger second-messenger signals thereby dictating an appropriate steering response. The biological activity of such receptors or ligands can be measured by overexpressing the receptor or hgand protein in culture and then monitoring growth cone guidance (Kremoser et al, 1995, Ceh 82: 359). Attraction or repulsion of cehs which is observed to be different than normal is an indication of the role of this protein in growth guidance, and identifies the polymorphisms as altering function.

x. Changes in gene expression or protein function that result from the presence of a polymorphism can be detected by in vivo assays including the production of transgenic animals, knock out animals or the analysis of naturally occurring animal models of a particular disease.

Transgenic Animals

Transgenic mice provide a useful tool for genetic and developmental biology studies and for the determination of a function of a novel sequence. According to the method of conventional transgenesis, additional copies of normal or modified genes are injected into the male pronucleus of the zygote and become integrated into the genomic DNA of the recipient mouse. The transgene is transmitted in a Mendehan manner in estabhshed transgenic strains.

Constructs useful for creating transgenic animals comprise genes under the control of either their normal promoters or an inducible promoter, reporter genes under the control of promoters to be analyzed with respect to their patterns of tissue expression and regulation, and constructs containing dominant mutations, mutant promoters, and artificial fusion genes to be studied with regard to their specific developmental outcome. Transgenic mice are useful according to the invention for analysis of the dominant effects of overexpressing a candidate gene in mouse. Typically, DNA fragments on the order of 10 kilobases or less are used to construct a transgenic animal (Reeves, 1998, New. Anat, 253:19). Transgenic animals can be created with a construct comprising a candidate gene containing one or more polymorphisms according to the invention. Alternatively, a transgenic animal expressing a candidate gene containing a single polymorphism can be crossed to a second transgenic animal expressing a candidate gene containing a different polymorphism and the combined effects of the two polymorphisms can be studied in the offspring animals. Transgenic mice engineered to overexpress a number of genes, including PCK1 (Valera et al., 1994, Proc. Natl. Acad. Sci. USA, 91: 9151), LNS (Mitanchez et al, FEBS Letters, 421: 285), IAPP (D'Alession et al, 1994, Diabetes, 43:1457), Asp (Klebig et al, Proc. Natl. Acad. Sci. USA, 92: 4728) and Agrt (Graham et al, Nature Genetics, 17:273), have been prepared and maybe useful for studying osteoarthritis.

Knock Out Animals

i. Standard

Knock out animals are produced by the method of creating gene deletions with homologous recombination. This technique is based on the development of embryonic stem (ES) cehs that are derived from embryos, are maintained in culture and have the capacity to participate in the development of every tissue in the mouse when introduced into a host blastocyst. A knock out animal is produced by dhecting homologous recombination to a specific target gene in the ES cehs, thereby producing a null ahele of the gene. The potential phenotypic consequences of this nuh ahele (either in heterozygous or homozygous offspring) can be analyzed (Reeves, supra). Single or double knock out mice that may be useful for studying osteoarthritis have been produced for a number of genes including IRS 1 (Araki et al, 1994, Nature, 372:186, Tamemoto et al, 1994, Natare, 372:182), 1R52 (Withers et al, 1998, Nature, 391:900), INSR, BIRKO, MIRKO, INSR (Lamothe et al, 1998, FEBS Letter, 426:381), GLUT2, GLUT4 (Katz et al, 1995, Natare, 377:151), GLPIR (Gahwitz and Schmidt, 1997, Z. Gastroenterol, 35:655):, GCK (Sakura et al, 1998, Diabetologia, 41:654), GCK/IRSl, IRSl INSR, MC4R (Huszar et al, 1997, Ceh, 88:13 1) and BRS3 (Ohki-Hamazaki et al, 1997, Natare, 390:165).

ii. In vivo Tissue Specific Knock Out in Mice Using Cre-lox.

The method of targeted homologous recombination has been improved by the development of a system for site-specific recombination based on the bacteriophage PI site specific recombinase Cre. The Cre-loxP site-specific DNA recombinase from bacteriophage PI is used in transgenic mouse assays in order to create gene knockouts restricted to defined tissues or developmental stages.

Regionally restricted genetic deletion, as opposed to global gene knockout, has the advantage that a phenotype can be attributed to a particular cell/tissue (Marth, 1996, Clin. Invest. 97: 1999). In the Cre- loxP system one transgenic mouse strain is engineered such that loxP sites flank one or more exons of the gene of interest. Homozygotes for this so cahed 'foxed gene' are crossed with a second transgenic mouse that expresses the Cre gene under control of a cell/tissue type transcriptional promoter. Cre protein then excises DNA between loxP recognition sequences and effectively removes target gene function (Sauer, 1998, Methods, 14:381). There are now many in vivo examples of this method, including the inducible inactivation of mammary tissue specific genes (Wagner et al, 1997, Nucleic Acids Res., 25:4323).

hi. Bac Rescue of Knock Out Phenotype

In order to verify that a particular genetic polymorphism/mutation is responsible for altered protein function in vivo one can "rescue" the altered protein function by introducing a wild-type copy of the gene in question. In vivo complementation with bacterial artificial chromosome (BAC) clones expressed in transgenic mice can be used for these purposes. This method has been used for the identification of the mouse circadian Clock gene (Antoch et al, 1997, Ceh 89: 655).

iv. Naturally Occurring Animal Models

Naturally occuning animal models useful for studying osteoarthritis include models of severe hyperglycaemia (celebes black ape, Chinese hamster, diabetes mouse (db), Djunjarian hamster, Egyptian sand rat, Hartley guinea pig, OLETF rat, New Zealand white rabbit, obese BBZ/Wor rat, rhesus monkey, South African hamster, spiny mouse), models for moderate hyperglycaemia (Cohen diabetic rat, GK rat, Japanese KK mouse, male Bristol CBA/Ca mouse, male eSS rat, male WKY fatty rat, male Wistar WBN/Kob rat, male ZDF rat, NZO mouse, obese mouse (ob), PBB/Ld mouse, spontaneously hypertensive corpulent (SHR/N-cp) rat, Tuco-tuco, Wehesley hybrid mouse, yehow obese mouse) and hnpahed glucose tolerance (ageing laboratory rats and mice, BHE rat, Fatty Zucker rat (fa), Mongolian gerbil, NON diabetic mouse, squirrel monkey, Yucatan miniature swine) (Pickup and Williams, eds., Textbook of Diabetes, 2nd Edition, Blackweh Science).

G. Production of an Amplified Product

Amphfied products useful according to the invention can be prepared by utihzing the method of PCR as described in Section B entitled "Production of a Polynucleotide Sequence Primers useful for producing an amphfied product according to the invention (e.g. an amphfied product comprising one or more polymorphisms) can be designed and synthesized as described in Section A entitled "Design and Synthesis of Ohgonucleotide Primers".

The invention provides methods (e.g. Southern blot analysis, PCR, primer extension and ohgonucleotide hybridization), of detecting a polymorphism in an amphfied product.

H. Production of a Mutant Protein

1. Expression of the Nucleotide Sequence In accordance with the present invention, polynucleotide sequences which encode candidate gene protein fragments, fusion proteins or functional equivalents thereof may be used in recombinant DNA molecules that direct the expression of a candidate gene protein in appropriate host cehs. Due to the inherent degeneracy of the genetic code, other DNA sequences which encode substantially the same or a functionahy equivalent amino acid sequence, may be used to clone and express the candidate gene protein. As wih be understood by those of skih in the art, it may be advantageous to produce candidate gene-encoding nucleotide sequences possessing non-naturahy occurring codons. Codons preferred by a particular prokaryotic or eukaryotic host (Munay et al, 1989, Nucleic Acid Res 17:477) can be selected, for example, to increase the rate of protein expression or to produce recombinant RNA transcripts having desirable properties, such as a longer hah-hfe as compared to transcripts produced from the naturally occuning sequence.

The nucleotide sequences of the present invention can be engineered in order to alter a candidate gene-encoding sequence for a variety of reasons, including but not limited to, alterations which modify the cloning, processing and/or expression of the gene product. For example, mutations may be introduced using techniques which are weh known in the art, e.g., site-directed mutagenesis to insert new restriction sites, to alter glycosylation patterns, to change codon preference or to produce splice^' variants. -_:

In another embodiment of the invention, a natural, modified or recombinant candidate gene protein-encoding sequence may be hgated to a. heterologous sequence to encode a fusion protein (as described in Section B entitled "Production of a Polynucleotide Sequence"). For example, for screening of peptide hbraries for inhibitors of candidate gene protein activity, it may be useful to encode a chimeric protein that is recognized by a commerciahy available antibody, a fusion protein may also be engineered to contain a cleavage site located between a candidate protein and the heterologous protein sequence, so that the protein of interest may be substantially purified away from the heterologous moiety fohowing cleavage.

In another embodiment of the invention, the sequence encoding the candidate gene protein may be synthesized, whole or in part, using chemical methods weh known in the art (see Caruthers, et al., 1980, Nuc Acids Res Symp Ser, 7:215, Horn, et al, 1980, Nuc Acids Res Symp Ser, 225, etc.) Alternatively, the protein itself, or a portion thereof, could be produced using chemical methods of synthesis. For example, peptide synthesis can be performed using various sohd-phase techniques

(Roberge, et al, 1995, Science, 269:202) and automated synthesis maybe achieved, for example, using the A.I. 431 A Peptide Synthesizer (Perkin Elmer) in accordance with the instructions provided by the manufacturer. The newly synthesized peptide can be substantially purified by preparative high performance hquid chromatography (e.g., Creighton, 1983, Proteins, Structures and Molecular Principles, WH Freeman and Co. New York NY). The composition of the synthetic peptides may be confirmed by amino acid analysis or sequencing (e.g., the Edman degradation procedure; Creighton, supra). Additionahy the amino acid sequence of interest, or any part thereof, may be altered during direct synthesis and/or combined using chemical methods with sequences from other proteins , or any part thereof, to produce a variant polypeptide.

2. Expression Systems In order to express a biologically active protein, the nucleotide sequence encoding the protein of interest or its functional equivalent, is inserted into an appropriate expression vector, i.e., a vector which contains the necessary elements for the transcription and translation of the inserted coding sequence.

Methods which are weh known to those skihed in the art can be used to construct expression vectors containing a protein-encoding sequence and appropriate transcriptional or translational controls. These methods include in vivo recombination or genetic recombination. Such techniques are described in Ausubel et al, supra and Sambrook et al, supra.

A variety of expression vector host systems may be utihzed to contain and express a protein product of a candidate gene according to the invention. These include but are not limited to microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid or cosmid DNA expression vectors; yeast transformed with yeast expression vectors; insect ceh systems infected with virus expression vectors (e.g., baculovirus); plant ceh systems transfected with virus expression vector (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with bacterial expression vectors (e.g., Ti or pBR322 plasmid); or animal ceh systems. The "control elements" or "regulatory sequences" of these systems vary in their strength and specificities and are those, nontranslated regions of the vector, enhancers, promoters, and 3' untranslated regions, which interact with host cehular proteins to cany out transcription and translation. Depending on the vector system and host utihzed, any number of suitable transcription and translation elements, including constitutive and inducible promoters, maybe used. For example, when cloning in bacterial systems, inducible promoters such as the hybrid lacZ promoter of the Bluescript® phagemid (Stratagene, LaJoha CA) or pSportl (Gibco BRL) and ptrp-lac hybrids and the like maybe used. The baculovirus polyhedron promoter may be used in insect cehs. Promoters or enhancers derived from the genomes of plant cehs (e.g., heat shock, RUBISCO; and storage protein genes) or from plant virus (e.g. viral promoters or leader sequences) may be cloned into the vector. In mammahan ceh systems promoters from the mammalian genes or from mammalian viruses are most appropriate. If it is necessary to generate a ceh line that contains multiple copies of the sequence encoding the protein product of the gene of interest, vectors based on 5V40 or EBV may be used with an appropriate selectable marker.

In bacterial systems, a number of expression vectors may be selected depending upon the use intended for the protein of interest. For example, when large quantities of a protein are required for the production of antibodies, vectors which direct high level expression of fusion proteins that are readily purified may be desirable. Such vectors include, but are not limited to, the multifunctional E. coli cloning and expression vectors such as Bluescript® (Stratagene), in which the sequence encoding the protein of interest may be hgated into the vector in frame with sequences encoding the ammo-terminal Met and the subsequent 27 residues of b-galactosidase so that a hybrid protein is produced; pIN vectors (Van Heeke & Schuster, 1989, J Biol Chem 264:5503); and the like. Pgex vectors (Promega, Madison WI) may also be used to express foreign polypeptides as fusion proteins with GST. In general, such fusion proteins are soluble and can easily be purified from lysed cehs by adsorption to glutathione-agarose beads fohowed by elution in the presence of free glutathione. Proteins made in such systems are designed to include heparmn, thrombin or factor XA protease cleavage sites so that the cloned polypeptide of interest can be released from the GST moiety at wih.

In the yeast, Saccharomyces cerevisiae, a number of vectors containing constitutive or inducible promoters such as alpha factor, alcohol oxidase and PGH may be used. For reviews, see Ausubel et al (supra) and Grant et al, 1987, Methods in Enzymology 153:516.

In cases where plant expression vectors are used, the expression of a sequence encoding a protein of interest may be driven by any of a number of promoters. For example, viral promoters such as the 35S and 19S promoters of CaMV (Brisson et al., 1984, Nature 310:511) maybe used alone or in combination with the omega leader sequence from TMV (Takamatsu et al, 1987, EMBO J 6:307). Alternatively, plant promoters such as the smah subunit of RUBISCO (Coruzzi et al, 1984, EMBO J 3:1671; Broghe et al, 1984, Science, 224:838); or heat shock promoters (Winter I and Sinibaldi RM, 1991, Results Probl Ceh Differ., 17:85) maybe used. These constructs can be introduced into plant cehs by direct DNA transformation or pathogen-mediated transection. For reviews of such techniques, see Hobbs S or Muny LE in McGraw Hih Yearbook of Science and Technology (1992) McGraw Hih New York NY, pp 191-196 or Weissbach and Weissbach (1988) Methods for Plant Molecular Biology, Academic Press, New York, pp 421-463.

An alternative expression system which could be used to express a protein of interest is an insect system. In one such system, Autographa califomica nuclear polyhedrosis virus (AcNPV) is used as a vector to express foreign genes in Spodoptera frugiperda cehs or in Trichoplusia larvae. The sequence encoding the protein of interest may be cloned into a nonessential region of the virus, such as the polyhedrin gene, and placed under control of the polyhedrin promoter. Successful insertion of the sequence encoding the protein of interest wih render the polyhedron gene inactive and produce recombinant virus lacking coat protein coat. The recombinant viruses are then used to infect S. fiigoerda cehs or Trichoplusia larvae in which the protein of interest is expressed (Smith et al, 1983., J Virol 46:584; Engelhard, et al, 1994, Proc Natl Acad Sci 91:3224).

In mammalian host cehs, a number of viral-based expression systems may be utihzed. In cases where an adenovirus is used as an expression vector, a sequence encoding the protein of interest may be hgated into an adenovirus transcription/translation complex consisting of the late promoter and tripartite leader sequence. Insertion in a nonessential El or E3 region of the viral genome wih result in a viable virus capable of expressing in infected host cehs (Logan and Sherik, 1984, Proc Natl Acad Sci, 81:3655). In addition, transcription enhancers, such as the rous sarcoma virus (RSV) enhancer, may be used to increase expression in mammalian host cehs.

Specific initiation signals may also be required for efficient translation of a sequence encoding the protein of interest. These signals include the ATG initiation codon and adjacent sequences. In cases where the sequence encoding the protein, its initiation codon and upstream sequences are inserted into the most appropriate expression vector, no additional translational control signals may be needed. However, in cases where only coding sequence, or a portion thereof, is inserted, exogenous transcriptional control signals including the ATG initiation codon must be provided. Furthermore, the initiation codon must be in the coπect reading frame to ensure transcription of the entire insert. Exogenous transcriptional elements and initiation codons can be of various origins, both natural and synthetic. The efficiency of expression may be enhanced by the inclusion of enhancers appropriate to the ceh system in use (Scharf, et al, 1994, Results Probl Ceh Differ, 20:125; Bittner et al, 1987, Methods in Enzymol, 153:516).

In addition, a host ceh strain may be chosen for its abihty to modulate the expression of the inserted sequences or to process the expressed protein in the deshed fashion. Such modifications of the polypeptide include but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, hpidation and acylation. Post-translational processing which cleaves a "prepro" form of the protein may also be important for correct insertion, folding and/or function. Different host cehs such as CHO, HeLa, MDCK, 293, W138, etc have specific cehular machinery and characteristic mechanisms for such post-translational activities and may be chosen to ensure the conect modification and processing of the introduced, foreign protein.

For long-term, high-yield production of recombinant proteins, stable expression is preferred. For example, ceh lines which stably express a foreign protein may be transformed using expression vectors which contain viral origins of replication or endogenous expression elements and a selectable marker gene. Fohowing the introduction of the vector, cehs may be ahowed to grow for 1-2 days in an enriched media before they are switched to selective media. The purpose of the selectable marker is to confer resistance to selection, and its presence ahows growth and recovery of cehs which successfully express the introduced sequences. Resistant clumps of stably transformed cehs can be expanded using tissue culture techniques appropriate to the ceh type. Any number of selection systems may be used to recover transformed ceh lines. These include, but are not limited to, the herpes simplex virus thymidine kinase (Wigler., et al, 1977, Ceh 11:223) and adenine phosphoribosyltransferase (Lowy, et al, 1980, Ceh 22:817) genes which can be employed in tk- or aprt- cehs, respectively. Also, antimetabolite, antibiotic or herbicide resistance can be used as the basis for selection; for example, dhfr which confers resistance to methotrexate (Wigler et al, 1980, Proc Natl Acad Sci 77:3567); npt, which confers resistance to the aminoglycosides neomycin and G-418 (Colbere-Garapin et al, 1981., J Mol Biol, 150:1) and als or pat, which confer resistance to cMorsulfuron and phosphinotricin acetyltransferase, respectively (Muny, supra). '

Additional selectable genes have been described, for example, trpB, which ahows cehs to utihze indole in place of tryptophan, or hisD, which ahows cehs to utihze histinol in place of histidine (Hartman and Mulligan, 1988, Proc Natl Acad Sci 85:8047). Recently, the use of visible markers has gained popularity with such markers as anthocyanins, B glucuronidase and its substrate, GUS, and luciferase and its substrate, luciferin, being widely used not only to identify transformants, but also to quantify the amount of transient or stable protein expression attributable to a specific vector system (Rhodes et al, 1995, Methods Mol Biol 55:121).

3. Identification of Transformants Containing the Polynucleotide Sequence

Although the presence/absence of marker gene expression suggests that the gene of interest is also present, its presence and expression should be confirmed. For example, if the sequence encoding a foreign protein is inserted within a marker gene sequence, recombinant cehs containing the sequence encoding the foreign protein can be identified by the absence of marker gene function.

Alternatively, a marker gene can be placed in tandem with the sequence encoding the foreign protein under the control of a single promoter. Expression of the marker gene in response to induction or selection usuahy indicates expression of the tandem sequences as weh. Alternatively, host cehs which contain the coding sequence for a protein of interest and express the protein of interest may be identified by a variety of procedures known to those of skih in the art. These procedures include, but are not limited to, DNA-DNA or DNA-RNA hybridization and protein bioassay or immunoassay techniques which include membrane, solution, or chip based technologies for the detection and/or quantification of the nucleic acid or protein.

The presence of the polynucleotide sequence encoding the protein of interest can be detected by DNA-DNA or DNA-RNA hybridization or amphfication using probes, portions or fragments of the sequence encoding the foreign protein of interest.

A variety of protocols for detecting and measuring the expression of the foreign protein, using either polyclonal or monoclonal antibodies specific for the protein are known in the art. Examples include enzyme-linked immunosorbant assay (ELISA), radioimmunoassay (RIA) and fluorescent activated ceh sorting (FACS). A two-site, monoclonal-based immunoassay utilizing monoclonal antibodies reactive to two non-interfering epitopes on the protein of interest is prefened, but a competitive binding assay may be employed. These and other assays are described in Hampton et al, 1990, Serological Methods a Lahoratory Manual, APS Presds, St Paul MN and Maddox., et al, 1983, J Exp Med 158:1211.

4. Purification of the Protein of Interest

Host cehs transformed with a nucleotide sequence encoding a protein of interest may be cultured under conditions suitable for the expression and recovery of the encoded protein from ceh culture. The protein produced by a recombinant ceh maybe secreted or contained intracehularly depending on the sequence and/or the vector used. As wih be understood by those of skih in the art, expression vectors containing a sequence encoding a protein of interest can be designed with signal sequences which direct secretion of the protein of interest through a prokaryotic or eucaryotic ceh membrane. Other recombinant constructions may join the sequence encoding the protein of interest to the nucleotide sequence encoding a polypeptide domain which wih facihtate purification of soluble proteins (Kroh et al, 1993, DNA Cell Biol, 12:441).

The protein of interest may also be expressed as a recombinant protein with one or more additional polypeptide domains added to facihtate protein purification. Such purification facilitating domains include, but are not limited to, metal chelating peptides such as a histidine-tryptophan modules that ahow purification on immobihzed metals, protein a domains that ahow purification on immobihzed immunoglobuhn, and the domain utihzed in the FLAGS extension/affinity purification system (Immunex Corp, Seattle WA). The inclusion of a cleavable linker sequences such as Factor XA or enterokinase (Invitrogen, San Diego CA), between the purification domain and the protein of interest is useful for facilitating purification. One such expression vector provides for expression of a fusion protein comprising the sequence encoding a foreign protein and nucleic acid sequence encoding 6 histidine residues fohowed by thioredoxin and an enterokinase cleavage site. The histidine residues facihtate purification while the enterokinase cleavage site provides a means for purifying the foreign protein from the fusion protein.

In addition to recombinant production, fragments of the protein of interest may be produced by direct peptide synthesis using sohd-phase techniques (Stewart et al, 1969, Solid-Phase Peptide Synthesis, WH Freeman Co,. San Francisco; Merrifield, 1963, J Am Chem Soc, 85:2149). In vitro protein synthesis may be performed using manual techniques or by automation. Automated synthesis maybe achieved, for example, using Apphed Biosystems 431 A Peptide Synthesizer (Perkin Elmer, Foster City CA) in accordance with the instructions provided by the manufacturer. Various fragments of a protein of interest may be chemically synthesized separately and combined using chemical methods to produce the full length molecule.

I. Preparation of Antibodies

Antibodies specific for the protein products of the candidate genes of the invention are useful for protein purification, for the diagnosis and treatment of various diseases (e.g osteoarthritis) and for drug screening and drug design methods useful for identifying and developing compounds to be used in the treatment of various diseases (e.g. osteoarthritis). By antibody, we include constmctions using the binding (variable) region of such an antibody, and other antibody modifications. Thus, an antibody useful in the invention may comprise a whole antibody, an antibody fragment, a polyfunctional antibody aggregate, or in general a substance comprising one or more specific binding sites from an antibody. The antibody fragment may be a fragment such as an Fv, Fab or F(ab')₂ fragment or a derivative thereof, such as a single chain Fv fragment. The antibody or antibody fragment maybe non- recombinant, recombinant or humanized. The antibody may be of an immunoglobulin isotype, e.g., IgG, lgM, and so forth. In addition, an aggregate, polymer, derivative and conjugate of an immunoglobulin or a fragment thereof can be used where appropriate. Neutralizing antibodies are especially useful according to the invention for diagnostics, therapeutics and methods of drug screening and drug design.

Although a protein product (or fragment or ohgopeptide thereof) of a candidate gene of the invention that is useful for the production of antibodies does not require biological activity, it must be antigenic. Peptides used to induce specific antibodies may have an amino acid sequence consisting of at least five amino acids and preferably at least 10 amino acids. Preferably, they should be identical to a region of the natural protein and may contain the entire amino acid sequence of a smah, naturally occurring molecule. Short stretches of amino acids corresponding to the protein product of a candidate gene of the invention maybe fused with amino acids from another protein such as keyhole hmpet hemocyanin or GST, and antibody wih be produced against the chimeric molecule. Procedures weh known in the art can be used for the production of antibodies to the protein products of the candidate genes of the invention.

For the production of antibodies, various hosts including goats, rabbits, rats, mice etc... maybe immunized by injection with the protein products (or any portion, fragment, or ohgonucleotide thereof which retains immunogenic properties) of the candidate genes of the invention. Depending on the host species, various adjuvants maybe used to increase the immunological response. Such adjuvants include but are not limited to Freund's, mineral gels such as aluminum hydroxide, and surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, and dinitrophenol. BCG (bacilli Calmette-Guerin) and Corynebacterium parvum are potentiahy useful human adjuvants .

1. Polyclonal antibodies.

The antigen protein may be conjugated to a conventional carrier in order to increase its immunogenicity, and an antiserum to the peptide-carrier conjugate wih be raised. Coupling of a peptide to a caπier protein and immunizations maybe performed as described (Dymecki et al, 1992, J. Biol. Chem., 267: 4815). The serum can be titered against protein antigen by ELISA (below) or alternatively by dot or spot blotting (Boersma and Van Leeuwen, 1994, J Neurosci. Methods, 51: 317). At the same time, the antiserum may be used in tissue sections prepared asdescribed. A useful serum wih react strongly with the appropriate peptides by ELISA, for example, fohowing the procedures of Green et al, 1982, Ceh, 28: 477.

2. Monoclonal antibodies.

Techniques for preparing monoclonal antibodies are weh known, and monoclonal antibodies may be prepared using a candidate antigen whose level is to be measured or which is to be either inactivated or affinity-purified, preferably bound to a carrier, as described by Arnheiter et al, 1981, Nature, 294;278.

Monoclonal antibodies are typically obtained from hybridoma tissue cultures or from ascites fluid obtained from animals into which the hybridoma tissue was introduced.

Monoclonal antibody-producing hybridomas (or polyclonal sera) can be screened for antibody binding to the target protein.

3. Antibody Detection Methods

Particularly preferred immunological tests rely on the use of either monoclonal or polyclonal antibodies and include enzyme-linked immunoassays (ELISA), immunoblotting and immunoprecipitation (see Voher, 1978, Diagnostic Horizons, 2:1, Microbiological Associates Quarterly Publication, Walkersville, MD; Voher et al, 1978, J. Clin. Pathol, 31: 507; U.S. Reissue Pat. No. 31,006; UK Patent 2,019,408; Butler, 1981, Methods Enzymol, 73: 482; Maggio, E. (ed.), 1980, Enzyme Immunoassay, CRC Press, Boca Raton, FL) or radioimmunoassays (RIA) (Weintraub, B., Principles of radioimmunoassays, Seventh Training Course on Radiohgand Assay Techniques, The Endocrine Society, March 1986, pp. 1-5, 46-49 and 68-78). For analysing tissues for the presence or absence of a protein produced by a candidate gene according to the present invention, imrnunohistochemistry techniques may be used. It wih be apparent to one skihed in the art that the antibody molecule may have to be labehed to facihtate easy detection of a target protein. Techniques for labelling antibody molecules are weh known to those skihed in the art (see Harlow and Lane, 1989, Antibodies, Cold Spring Harbor Laboratory).

J. Preparation of a Labeled Protein

1. Lab ng of protein

Labeling techniques are useful, according to the invention, for studying the biochemical properties, processing, intracehular transport, secretion and degradation of proteins.

Biosynthetic labeling of proteins produced by candidate genes of the invention is preferably performed with ³⁵S -methionine due to the high specific activity (>800Ci/mmol) and ease of detection of this amino acid. Another amino acid should be used to label a protein that contains little or no methionine.

According to the fohowing protocol, either suspension cehs or adherent cehs are labeled with ^3SS-methionine. Briefly, cehs are washed and incubated for 15 min at 37°C in short-term labeling medium (complete serum-free, melhionine free RPMI or DMEM containing 5% (v/v) dialyzed fetal bovine serum) to deplete intracehular pools of met onine. Cehs are then incubated in the presence of ³⁵S-me1hionine working solution (0.1 to 0.2 mCi/ml in 37°C short-term labeling medium) such that 4ml of ³⁵S-me1hioιιine working solution is added per 2 x 10⁷ suspension cehs and 2 to 4 ml of ³⁵S- metmonine working solution is added per 100 mm dish of adherent cehs (0.5-2 x 10⁷ cehs), for a period of 30 min to 3 hour in a humidified, 37°C, 5% C0₂ incubator. Upon completion of labeling, suspension cehs are washed by centrifugation in ice-cold PBS. Fohowing removal of labeling medium, adherent cehs are washed with PBS, scraped and cohected by centrifugation. Labeled cehs are processed and analyzed by immuno affinity chromatography, immunoprecipitation and one- and two-dimensional gel electrophoresis (Ausubel et al, supra).

If the protein of interest is synthesized at a relatively low rate or is in a steady state, it may be necessary to label cehs for an extended period of time. When performing long-term biosynthetic labeling of cehs, it is necessary to include unlabeled methionine in the medium to maintain ceh viability and to ensure that incorporation of label is maintained during the course of the experiment. According to this method, cehs can be labeled in the presence of ³⁵S-metMonine in long term labeling medium (90% methionine free RPMI or DMEM) for up to 16 hours (Ausubel et al, supra).

2. In vitro Translation The protein product of the cloned candidate gene of the invention can be produced by the methods of in vitro transcription and in vitro translation. In vitro transcription is performed essentiahy. as described in Section B entitled "Production of a Polynucleotide Sequence" in the absence of a labeled ribonucleoside. The RNA produced by the in vitro transcription reaction wih be extracted with phenol, ethanol precipitated twice and resuspended in 10ml of TE buffer. In vitro translation is performed by adding 1 to 10ml of RNA to an in vitro translation kit (e.g. wheat germ or reticulocyte lysate) in the presence of 15mCi [³⁵S]methionine, fohowing the directions provided by the manufacturer. A typical reaction is carried out in a 30ml volume at room temperature for 30 to 60 minutes (Ausubel et al, supra).

K. Production of Cells Expressing a Nucleotide Sequence Comprising a Polymorphism

Mammalian cehs expressing a nucleotide sequence comprising a polymorphism are useful, according to the invention for deterrnining the biochemical and functional properties of the protein product of a nucleotide sequence comprising a polymorphism, for analyzing expression of a candidate gene, for large scale production of a protein of interest, for drag screening and for the production of transgenic animals or knockout mice.

Methods of efficiently introducing foreign DNA into mammalian cehs are known in the art and include calcium phosphate transfection, DEAE-dextran transfection, electroporation and hposome- mediated transfection (Ausubel et al, supra).

Transfection Protocols

1. Calcium-Phosphate Transfection The method of calcium phosphate transfection involves preparing a precipitate by slowly mixing a HEPES -buffered saline solution with a mixtare of calcium chloride and DNA. According to this method, up to 10% of the cehs on a dish wih incorporate DNA.

Cehs to be transfected are spht one day prior to transfection so that on the day of transfection cehs are well-separated on the plate, a 10 cm dish of cehs is fed with 9.0 ml of complete medium approximately 2 to 4 hours before the addition of the precipitate. DNA to be transfected (10-50mg/10- c plate) is ethanol precipitated, resuspended in 450 ml sterile water and mixed with 50 ml of 2.5 M CaCl₂. The DNA/CaCl_j solution is added dropwise to a 15-rnl conical tube containing 500 ml 2X HeBS (0.283M NaCl, 0.023M HEPES acid, 1.5 mM Na^O^ pH 7.05). It is preferable to bubble the HeBS solution during the addition of the DNA mixture. After the precipitate has formed for 20 minutes at room temperatare, it is added evenly to the cehs. The cehs are incubated with the precipitate at 37°C in a C0₂ humidified incubator for 4-16 hours. Fohowing removal of the precipitate, the cehs are washed with PBS and fed in complete medium. Glycerol or dimethyl sulfoxide shock can ' be used to increase the DNA uptake by certain types of cehs (Ausubel et al, supra).

2. DEAE-Dextran Transfection

Cehs to be transfected are plated at a concentration such that after 3 days of growth they are 30-50% confluent. The DNA to be transfected (approximately 4 mg) is ethanol precipitated, resuspended in 40ml TBS and added slowly while shaking to 80 ml of warm 10 mg/ml DEAE-dextran in TBS. After cehs have been washed with PBS and fed with 4 ml of DMEM containing 10% Nu Serum/lOcm dish, the DEAE-dexfranlDNA mixtare is evenly distributed over the entire plate. Cehs are incubated with the DNA for approximately 4 hours in a humidified C0₂ incubator. Fohowing the removal of the DEAE-dextran/DNA mixtare, cehs are shocked by the addition of 5 ml of 10% DMSO in PBS. After a 1 minute incubation at room temperatare, cehs are washed with PBS and fed with complete medium (Ausubel et al., supra).

3. Electroporation

Alternatively, DNA can be introduced into cehs by the use of high-voltage electric shocks, a technique termed electroporation. Briefly, according to the method of electroporation, cehs are suspended in an appropriate electroporation buffer and placed in an electroporation cuvette. Fohowing the addition of DNA, the cuvette is connected to a power supply and the cehs are subjected to a high- voltage electrical pulse of a defined magnitude and length, optimized for the ceh type being > transfected. After a brief period of recovery, the cells are placed in normal culture medium. A population of cehs to be transfected by electroporation is grown to late-log phase in complete medium. Typically stable transfection requires 5 X 106 cehs, and transient transfection requires 1-4 X 10⁷ cehs. Cehs are harvested by centrifugation for 5 minutes at 640 x g at 4°C. The resulting ceh pehet is resuspended in hah of the original volume of ice-cold electroporation buffer (e.g. PBS without calcium or magnesium, Hepes buffered saline, tissue culture medium without serum, or phosphate buffered sucrose (272mM sucrose/7 mM K₂HP0₄, pH 7.4/lmM MgCl₂)). The choice of an electroporation buffer is dictated by the ceh line. Cehs are then harvested by centrifugation for 5 minutes at 640 x g at 4°C, and resuspended at 1 X 10⁷/ml in electroporation buffer at 0°C for stable transfection or at a higher concentration (up to 8 X 10⁷/ml) for transient transfection. Ahquots of the cehs (0.5 ml) are transferred into the deshed number of electroporation cuvettes and placed on ice. DNA is added to the ceh suspension in the cuvettes on ice. For stable transfection, DNA

(optimally 1-10 mg) should be linearized with a restriction enzyme that cuts at a site in a non-essential region, purified by phenol extraction and etlianol precipitated. Supercoiled DNA (optimally 10 mg) may be used for transient transfection. The DNA/ceh suspension is mixed, and incubated on ice for 5 minutes. The cuvette is placed in the holder in the electroporation apparatus (at room temperatare) and shocked one or more times at the deshed voltage and capacitance settings. An electroporation apparatus useful according to the invention is the Bio-Rad Gene Pulser. The number of shocks and the voltage and capacitance settings wih vary depending on the ceh type, and should be optimized. The two parameters that are critical for successful electroporation are the maximum voltage for the shock and the duration of the cunent pulse.

Fohowing electroporation, the cuvette containing the mixtare of cehs and DNA is incubated on ice for 10 minutes. The transfected cehs are diluted 20-fold in complete culture medium. For stable transfection cehs are grown for 48 hours in nonselective medium and then transfened to antibiotic containing medium. For transient transfection, cehs are incubated 50-60 hours and then harvested for the deshed transient assay.

L. Production of Animals Expressing a Nucleotide Sequence Comprising a Polymorphism Transgenic animals expressing a construct comprising a candidate gene containing a polymorphism, according to the invention can be produced by methods weh known in the art (reviewed in Reeves et al, supra). Knock out mice wherein a candidate gene according to the invention has been disrupted can be produced by methods weh known in the art (reviewed in Moreadifh and Radford, 1997, J,Mol Med., 75:208 and Shastry, 1998, Mol. Ceh. Biochem., 181:163). These animals provide useful models for studying the functional consequences of one or more polymorphisms in a gene of interest.

M. Production of a Candidate Gene Library The invention provides a method of producing a candidate gene hbrary comprising genes that are potentiahy associated with the susceptibihty to, or pathogenesis of a disease. A candidate gene hbrary is useful for determining the genetic basis of a disease of interest.

Genetic susceptibihty to a disease must occur as a result of specific DNA differences relative to non-susceptible individuals. In the case of osteoarthritis, many genes are known which are potentiahy involved in the susceptibihty to, or pathogenesis of the disease. These genes are included in the candidate gene hbrary and the association of these genes with osteoarthritis is determined from population studies according to the invention. Unlike linkage studies. wherein a region of the genome' that is. thought to be involved in a disease is determined, the candidate gene strategy, including association studies, addresses the involvement of a particular gene in a disease. The results of association studies of candidate genes are used to identify genes that should be intensively studied as potential therapeutics or therapeutic targets.

According to the invention, the full range of polymorphic sites within each candidate gene is identified and examined in diseased and normal populations. The frequency of each gene variant (ahele) in each population is then compared to the other. If a specific polymorphism under analysis contributes to the disease phenotype, it wih be present in the diseased population at a higher frequency than in the normal population. In addition, if the specific polymorphism under analysis does not itself contribute to the disease phenotype but resides elsewhere in, or is near to a gene containing a contributory polymorphism, a significant association maybe seen with the polymorphic marker being tested. This is because the two markers are in linkage disequihbrium with each other due to their close proximity.

1. Strategies for Identifying Genes Associated with a Disease

There are a number of methods known in the art for the identification of genes involved in a disease. These methods include familial linkage studies fohowed by positional cloning, differential gene expression studies on tissues, and population-based candidate gene association studies. Although positional cloning has proven to be useful for diseases resulting from a single mutation, this technique is not suitable for identifying genetic linkage in diseases where multiple genetic variants combine to create disease susceptibihty. Furthermore, it has been demonstrated that the etiological basis of the majority of diseases comprises more than one gene.

The goal of linkage studies is to determine the approximate position of disease genes by studying related individuals in famihes. According to linkage strategies, DNA markers that are randomly spaced throughout the genome, but are rarely located within genes, are tested for the frequency of their presence along with the particular disease phenotype. There is approximately a 50% chance of an unlinked gene and marker gene co-localizing. If a particular marker is present at a significantly higher frequency than expected in disease individuals, this indicates that the marker is located in the vicinity of the disease gene. Usuahy the disease gene is delimited to a large region (containing tens to hundreds of genes). After a disease gene has been grossly mapped, this entire region must be extensively characterized to determine what genes are present in the region. Any gene that is identified according to this method becomes a candidate gene.

Linkage studies have been used successfully to identify the genes responsible for certain genetic, diseases originating from mutations in a single gene (monogenic diseases). However, most common human diseases are of polygenic origin wherein changes in multiple genes causes an increased susceptibihty to or pathogenesis of a particular disease. Because the DNA changes associated with genes which contribute to polygenic diseases are common in the population, thereby diluting the contribution of a given region of the genome to the disease, it is difficult to perform linkage studies on diseases of polygenic origin.

Linkage analysis

A series of genetic crosses is performed in an animal model system of a particular defect that is characteristic of a disease of interest (e.g. osteoarthritis) between individuals having an observable mutant phenotype and normal individuals of a control strain. At least one disease- related loci is used as a marker in these crosses. Alternatively, linkage analysis ban be performed using chromosomal markers that do not comprise a disease related locus (described below). If non-random assortment of the mutant trait with a marker locus is observed, and if that non-random assortment is statisticahy significant (for example, if a Student's t test or ANOVA is apphed to the results) the trait is linked to the marker locus. Similarly, linkage analysis using an existing human or other mammalian pedigree may be performed. Pedigree analysis is a useful technique for identifying genes for which variant aheles may contribute to the risk, onset or progression of a disease in a family containing multiple individuals afflicted with a disease; according to this method, numerous genetic loci from affected and unaffected family members are compared. Non-random assortment of a given genetic marker between affected and unaffected family members relative to the distributions observed for other genetic loci indicates that the marker (for example, a variant isoform of a gene) either contributes to the disease or is in physical proximity to another that does so.

If a non-random assortment of the disease-related phenotype with a marker locus is observed, using either approach, this is indicative of an association between the gene underlying the defect and that locus. Because the strength of any conclusion drawn from linkage analysis is statisticahy-based, the accuracy of the results is thought to be proportional to the number of crosses or family members and genetic loci analyzed.

Positional Cloning

If linkage is confirmed it is preferable to perform a molecular analysis of the region in which the peak of linkage maps. The wide availability of yeast artificial chromosome (YAC) or bacterial artificial chromosome (BAC) hbraries facihtates this analysis, a nucleic acid sequence specific for a region encompassing a gene which is determined to occupy a map location of a particular locus of interest is examined, and open reading frames are evaluated to determine their relationship with the observed phenotype. An initial evaluation may be performed with the assistance of a computer program, such as the PathCalhng^1M(CuraGen) biological pathway discovery platform. Ah or a subset of the open reading frames present in the region are then cloned (e.g., by PCR) from mutant animals or affected family members and from their healthy counterparts (either control animals or unaffected family members), and the sequences of these open reading frames are compared. If a mutation or other ahehc variant is found to be linked to individuals displaying the disease phenotype (in a statisticahy-significant, non-random manner), it can be concluded that this mutation is associated with a disease phenotype. A nucleic acid fragment containing this gene can be labeled and used as a probe for in situ hybridization analysis of fixed chromosomes of the human or other mammal to determine precisely the physical location of the gene. Furthermore, a gene that has been mapped and isolated in this manner maybe useful as a candidate target for disease diagnosis and for drag targeting according to the invention (see below). 2. Identification of Genes to be Included in Candidate Gene Library

A candidate gene hbrary according to the invention wih include i. genes that are involved in known or predicted disease pathways, ii. new genes that are identified by a relevant pattern of specific tissue or ceh expression, hi. genes that map to genomic regions of known linkage, and iv. gene sequences (from sequence databases) that are homologs of the above referenced categories of potential candidate genes. The choice of potentiahy related genes to be selected from a database wih depend on the percent identity as calculated by Fast DB and based upon mismatch penalty, gap penalty, gap size penalty and joining penalty. Figure 1 summarized

Based on the physiological changes associated with a disease of interest, predictions can be made regarding a ceh or tissue-type that would be expected to express high or low levels of candidate genes associated with a particular disease. For osteoarthritis, it is expected that muscle, adipose, pancreas or hver tissue or tissue comprising insulin secreting pancreatic b-cehs, would be useful for identifying candidate genes according to the invention.

Differences in the expression of known and unknown genes in normal and disease tissue can be determined by methods known in the art including Serial Analysis of Gene Expression (SAGE) (Velcuescu et al, 1995, Science, 270:484), subtractive hybridization/screening (described below), differential display (Ling. and Pardee, 1992, Science, 257:967) high-density microaπay expression testing.

The technique of SAGE ahows for the rapid, detailed analysis of thousands of transcripts. SAGE depends on the fohowing two principles. Fhst, sufficient information is contained within a short nucleotide sequence (approximately 9-lObp), isolated from a defined location within a transcript, to uniquely identify a transcript. Second, the concatenation of short tags of sequence ahows transcripts to be analyzed serially by sequencing multiple tags within a single clone.

The method of SAGE is performed by synthesizing double-stranded cDNA from mRNA, cleaving the resulting cDNA with an anchoring restriction endonuclease that is expected to cleave most transcripts at least one time, and isolating the most 3 ' region of the cleaved cDNA by binding to streptavadin beads. This protocol ahows for the identification of a unique site on a transcript that conesponds to the restriction site located closest to the polyA tail. Replicate samples of the most 3' region of the cDNA are hgated to one of two linker molecules that contain a type US restriction site for a tagging enzyme. The cleavage site for Type IIS restriction endonucleases is located at a defined distance up to 20 bp from the asymmetric recognition site. Linkers are designed such that upon cleavage of the hgation product with the tagging enzyme there is release of the linker and an attached short region of cDNA. Fohowing the creation of blunt ends, the two pools of released tags are hgated to each other and the resulting hgated product is used as a template for PCR amphfication in the presence of primers that are specific for each linker. The PCR product is cleaved with the anchoring enzyme and amphfication products, comprising two tags linked tail to tail, are isolated, concatenated by hgation, cloned and sequenced (Velescu et al, supra).

Differential display provides a method for separating and cloning individual mRNAs by PCR analysis. According to the method of differential display, ohgonucleotide primers are selected wherein one primer is anchored to the polyadenylate tail of a subset of mRNA species and the other primer is short and of an arbitrary sequence such that it anneals at different positions relative to the first primer. The mRNA subpopulations that are identified with these primer pahs are subjected to reverse transcription, amphfied and analyzed on a DNA sequencing gel. By using multiple sets of primers, a reproducible pattern of amphfied cDNA fragments that demonstrate a requirement for the sequence specificity of either primer can be obtained (Liang and Pardee, supra).

According to the method of high-density microaπay expression testing, DNA sequences to be tested for expression are spotted onto a surface, usuahy at high-density to ahow for the testing of many genes. The surface contain the DNA sequences is typically referred to as a 'chip'. The spotted , DNA cam be either cDNA clones or ohgonucleotides. RNA is prepared from the two cehs or tissues , to be compared. The RNA from one cell/tissue wih be labeled red and the RNA from the other cell tissue wih be labeled yellow. Both RNA preparations are hybridized to the DNA anay. The ratio of red to yehow is indicative of the relative levels of expression between the two cells/tissues.

3. Mapping a candidate gene

Molecular and cytogenetic methods of mapping candidate genes are known in the art and are summarized below. Linkage analysis provides a method for identifying genes mapping to genomic regions of known linkage.

Linkage analysis

As described above, linkage analysis may be performed between an unmapped candidate gene and one or more of the disease-related loci or by analyzing the genetic linkage between the candidate gene and chromosomal markers which are not themselves linked to a disease-related locus, according to the same method. For the latter type of analysis it is preferable that the spacing of markers throughout the genome of the test organism is approximately one every cM or less. This spacing wih ensure complete coverage of the genome and wih facihtate accurate mapping.

Ill Other methods for mapping a candidate gene are provided below.

Syntenic similarity

As a result of classical genetic studies and, more recently, multi-laboratory genomic sequencing collaborations such as the Human Genome Project and Mouse Genome Project, the human and mouse genomes have been extensively characterized. It is now known that there is a significant degree of co-linearity among human, mice and rats wherein there is conservation relative to one another among these several species in the chromosomal map positions of numerous genes and groups of genes. Examination of the human and/or mouse chromosomal maps in the regions comparable to those to which a particular loci of interest maps in the rat wih yield candidate genes which may be responsible for the physiological changes associated with a disease of interest. The methods of radiation hybrid mapping or fluorescence in situ hybridization at low stringency to rat chromosomes using labeled fragments derived from the human or mouse genes can be used to confirm that genes present in these regions of the human and/or mouse are present in the regions of interest in the rat.

Radiation hybrid (RH) mapping is a somatic ceh hybrid technique that was developed to Greate high resolution, contiguous maps of mammalian chromosomes. The method is useful for , ordering DNA markers spanning millions of base pahs of DNA at a resolution not easily obtained by other mapping methods (Cox et al, 1990, Science, 250: 245; Burmeister et al, 1991, Genomics, 9:19; Waπington et al, 1992, Genomics, 13: 803; Abel et al., 1993, Genomics, 17:632). Radiation hybrid mapping facihtates the mapping of non-polymorphic DNA markers that cannot be used for meiotic mapping.

According to the method of radiation hybrid mapping a lethal dose of X-irradiation is used to fragment the chromosomes of the donor ceh line. Chromosome fragments from the donor ceh line are then retained, in a non-selective manner, fohowing ceh fusion with a recipient ceh line. The resulting hybrid clones are then analyzed for the presence or absence of specific donor chromosome markers. It is expected that markers that are further apart on a chromosome are more likely to be broken apart by radiation and to segregate independently in the RH cehs than markers that are closer together. By performing a statistical analysis of the co-segregation of various loci in hybrid clones, it is possible to construct a map that provides hrformation regarding the relative order and distance of markers (Cox et al., 1990, supra; Warrington et al, 1991, Genomics, 11: 701; Ceccherini et al, 1992, Proc. Natl. Acad. Sci. USA, 89: 104). Subtractive screening

In view of the observation that only a subset of an organism's genes are expressed in a given tissue, there is a high probabihty that transcripts which differ in expression between cehs of the same tissue in a mutant and control animal are responsible for the observed mutant phenotype. According to the method of subtractive cloning, mRNA is isolated from a tissue of choice, wherein the tissue is obtained from two distinct organisms and wherein one organism displays a mutant phenotype with regard to a particular trait while the other is normal in that respect. Methods weh known in the art are used to prepare cDNA from the mRNA derived from the organism. The mRNA template is then degraded, either by hydrolysis under alkaline conditions or by RNAase H- mediated cleavage, and the cDNA is returned to a buffer in which mRNA is stable, and mixed with a molar excess of mRNA prepared from the second organism under conditions of stringent hybridization. The mixture is then passed over a hydroxyapatite column, which binds double-stranded nucleic acids but ahows single stranded nucleic acid molecules to pass through. Reverse transcripts derived from the first sample which do not hybridize to niRNA molecules derived from the second organism (in other words, reverse transcripts specific to the first tissue sample) are present in the flow-through fraction and are cloned into a vector to create a subtraction hbrary. The reciprocal experiment (in which the cDNA is derived from the second mRNA preparation) is also caπied out to ' create a complete set of transcripts specific to the tissue samples derived from the two organisms. This procedure wih provide transcripts that can be labeled and used as probes in in situ hybridization analysis of immobihzed chromosomes. The method of subtractive screening therefore, yields both cloned genes as weh as reagents useful for determining if the cloned genes co-localize with a loci of interest. If a particular gene is found to co-localize to a loci of interest, the genes may be analyzed functionally (e.g., in a phenotypic rescue experiment, as described below or by the phenotypic assays described in Section F entitled "Identification and Characterization of Polymorphisms") Ultimately, these genes may be used as targets for drugs or disease diagnostic methods, or even as therapeutic nucleic acids.

Mutagenic transposon mapping

The selection of insertional events that he within genes (e.g., wi in coding or regulatory sequences) is facihtated by the use of entrapment vectors, first described in bacteria (Casadaban and Cohen, 1979, Proc. Natl. Acad. Sci. U.S.A., 76: 4530; Casadaban et al, 1980, J Bacteriol, 143: 971). By employing animal models, entrapment vectors can be introduced into pluripotent ES cehs in culture (for example, using electroporation or a retrovirus) and then passed into the germline via chimeras (Gossler et al, 1989, Science, 244: 463; Skames, 1990, Biotechnology, 8:827). Alternatively, transgenic animals containing entrapment vectors maybe generated by standard oocyte injection protocols.

These methods result in DNA integrations that are highly mutagenic because they interrupt the endogenous coding sequence. It is estimated that the frequency of obtaining a mutation in some gene of any in the genome using a promoter or gene trap is about 45%. For adetailed description of retrovhal insertion mutagenesis see Methods Enzymol, vol. 225, 1990. Genes which are expressed in a tissue of interest and for which a biochemical assay of a particular activity have been developed in animal models are most useful according to this method. Promoter or gene trap vectors often contain a reporter gene, e.g., lacZ, Cat or green fluorescent protein (Gfp) that lacks its own upstream promoter and/or sphce acceptor sequence. That is, promoter gene traps contain a reporter gene with a sphce site but no promoter. If the vector integrates within a gene and is sphced into the gene product, then the reporter gene wih be expressed. Enhancer traps contain a reporter gene and have a minimal promoter which requires the activity of an enhancer in order to function. If the vector integrates near an enhancer (whether in a gene or not), then the reporter gene wih be expressed. Activation of the reporter gene can only occur when the vector is integrated within an active host gene and generates a fusion transcript with the host gene. The activity of a reporter gene provides an easy assay for determining if a vector has been integrated into an expressed gene. Methods for detecting reporter gene activity in transfected cehs or tissues of a transgenic animal are weh known in the art.

The mutagenic vector may be mapped using standard cytogenetic techniques, such as in situ hybridization, wherein a labeled fragment comprising vector-specific sequence is used as a probe. Co- localization of the probe with a particular locus of interest indicates that the associated gene is a suitable candidate and should be subjected to further analysis. A gene that has been identified in this manner can be cloned as described.

N. Diagnostic Indicators, Screens and Disease Symptoms

In another embodiment of the invention, there is provided a method of diagnosing or determining susceptibihty of a subject to joint space narrowing and/or osteophyte development and/or joint pain. This method involves analyzing the genetic material of a subject to deterrnine which, allele(s) of a gene is/are present. The method may include detemώhng whether one or more particular aheles are present, or which combination of aheles (i.e. a haplotype) is present. The method may also include deteπriining whether subjects are homozygous or heterozygous for a particular ahele or haplotype.

In a preferred embodiment, the method comprises determining which allele of one or more polymorphisms of the invention is/are present. In particulai-, the method may include determining the presence of a polymorphism of a gene which in combination with polymoi hisrns defined herein or other polymorphisms may define a risk haplotype. The polynucleotides sequences for tliese particular alleles may be used for diagnostic purposes. The polynucleotides which may be used include ohgonucleotides, complementary RNA and DNA molecules and PNAs. The polynucleotides may be used to determine whether subjects are homozygous or heterozygous for a particular ahele or haplotype making them susceptible to joint space narrowing and/or osteophyte development and/or joint pain, and hence, osteoarthritis.

In one aspect, hybridization with a PCR probe which is capable of detecting a particular polymorphism may be used to identify nucleic acid sequences of particular aheles or haplotype. These probes must be specific to these particulai- aheles and the stringency of the hybridization or amplification must be such that the probe identifies only this particular ahele.

Means for producing specific hybridization probes for these polynucleotides of particular alleles include the cloning of these polynucleotide sequences into vectors for the production of mRNA probes is weh known to one skilled in the art. Such vectors are known in the art, are commerciahy available, and may be used to synthesize RNA probes in vitro by means of the addition of the appropriate RNA polymerases and the appropriate labeled nucleotides. Hybridization probes may be labeled by a variety of reporter groups, for example, by radionuchdes such as ³²P or ³⁵S, or by enzymatic labels, such as alkaline phosphatase coupled to the probe via avidin/biotin coupling systems, and the like.

Polynucleotides of particular alleles or haplotype may be used in Southern or northern analysis, dot blot, or other membrane-based technologies; in PCR technologies; in dipstick, pin, and multiformat ELISA-like assays; and in microarrays utilizing fluids or tissues from patients to detect susceptibihty to joint space narrowing and/or osteophyte development and/or joint pain. Such qualitative methods are weh known in the art.

In a particular embodiment, polynucleotides of particular aheles or haplotype may be used in assays that detect susceptibihty to joint space narrowing and/or osteophyte development and/or joint pain, particularly those mentioned above. Polynucleotides complementary to sequences of a particular ahele or haplotype may be labeled by standard methods and added to a fluid or tissue sample from a patient under conditions suitable for the formation of hybridization complexes. After a suitable incubation period, the sample is washed and it is deteπnined if there is a signal. If a signal is found, then the presence of the polynucleotide of a particular ahele, aheles or haplotype in the sample indicates the susceptibihty to joint space narrowing and/or osteophyte development and/or joint pain, and hence, osteoarthritis. Such assays may also be used to detemiine the particular therapeutic treatment regimen for an individual patient.

With respect to osteoarthritis, the presence of a particular polymorphism or polymorphisms in a tissue sample from an individual may indicate a predisposition for joint space narrowing and/or osteophyte development and/or joint pain, or may provide a means for detecting osteoarthritis prior to the appearance of actual clinical symptoms. A more definitive diagnosis of this type may ahow health professionals to employ preventative measures or aggressive treatment earlier, thereby preventing the development or further progression of osteoarthritis.

Additional diagnostic uses for ohgonucleotides designed from the polynucleotide sequences of a particular ahele or haplotype may involve the use of PCR. These ohgomers may be chemically synthesized, generated enzymaticahy, or produced in vitro. Ohgomers will contain a fragment of a polynucleotide a particular ahele, aheles or haplotype or a fragment of a polynucleotide complementary to the polynucleotide a particular allele, aheles or haplotype, and will be employed under optimized conditions for identification of a specific polymorphism, polvmo hisms or haplotype. Ohgomers may also be employed under very stringent conditions for detection of these particular DNA or RNA sequences. In further embodiments, ohgonucleotides or longer fragments derived from any of the polynucleotides described herein may be used as elements on a micro array. The micro array can be used in transcript imaging techniques to detect a particular polymorphism, polymorphisms or haplotype simultaneously as described below. In particular, this information may be used to develop a pharmacogenomic profile of a patient in order to select the most appropriate and effective treatment regimen for that patient. For example, therapeutic agents which are highly effective and display the fewest side effects may be selected for a patient based on his/her pharmacogenomic profile.

Microarrays may be prepared, used, and analyzed using methods known in the art (Brennan, T.M. et al. (1995) U.S. Patent No. 5,474,796; Schena, M. et al. (1996) Proc. Natl. Acad. Sci. USA 93:10614-10619; Baldeschwefler et al. (1995) PCT apphcation WO95/251116; Shalon, D. et al. (1995) PCT apphcation WO95/35505; Heller, R.A. et al. (1997) Proc. Natl. Acad. Sci. USA 94:2150-2155; Heller, M . et al. (1997) U.S. Patent No. 5,605,662). Various types of microarrays are weh known and thoroughly described in Schena, M., ed. (1999; DNA Micro aιτays: A Practical Approach, Oxford University Press, London). In another embodiment, a method involves the use of antibodies in diagnosing or determining the susceptibflity to joint space narrowing and/or osteophyte development and/or joint pain. The antibodies would specificaUy bind to an epitope of a particular ahele or form of the protein and may be used to determine susceptibihty to joint space narrowing and/or osteophyte development and/or joint pain, and hence, osteoarthritis. Antibodies useful for diagnostic purposes may be prepared in the same manner as described above. Diagnostic assays for deteπriining susceptibihty to joint space narrowing and/or osteophyte development and/or joint pain include methods which utihze the antibody and a label to detect a particular aflele or form of the protein in human body fluids or in extracts of ceUs or tissues. The antibodies may be used with or without modification, and may be labeled by covalent or non-covalent attachment of a reporter molecule. A wide variety of reporter molecules are known in the art and may be used.

A variety of protocols for measuring a particular allele or form of the protein, including- ELISAs, RIAs, and FACS, are known in the art and provide a basis for diagnosing susceptibflity to joint space narrowing and/or osteophyte development and/or joint pain.

O. Preparation of a Human Sample

The presence of an ahehc form of a gene containing a sequence variation, according to the invention, can be detected by testing any tissue of a human subject. Human samples that are useful according to the invention include tissue or fluid samples containing a polynucleotide or polypeptide of interest, include but are not limited to plasma, serum, spinal fluid, lymph fluid, urine, stool, external secretions of the skin, respiratory, intestinal and genitoruinary tracts, sahva, blood cehs, tamors, organs, tissue and samples of in vitro ceh culture constituents. Genomic DNA, cDNA or RNA can be prepared from the human sample according to the methods described above.

P. Methods of Use 1. Nucleic Acid Diagnosis and Diagnostic Kits

In order to detect the presence of an ahele of a gene predisposing an individual to osteoarthritis, a biological sample such as blood is prepared and analyzed for the presence or absence of susceptibihty aheles of a gene containing a polymorphism, according to the invention. Results of. these tests and interpretive information wih be returned to the health care provider for communication to the tested individual. Such diagnoses may be performed by diagnostic laboratories, or, alternatively, diagnostic kits are manufactured and sold to health care providers or to private individuals for self- diagnosis.

Initiahy, the screening method wih involve amphfication of the relevant gene sequences. In another prefened embodiment of the invention, the screening method involves a non-PCR based strategy. Such non-PCR based screening methods include Southern blot analysis to detect the presence of a variant form of a gene in a sample comprising total genomic DNA from the individual being tested. Alternatively, northern blot analysis can be used to detect an aberrant mRNA encoded by a gene, that exhibits altered stabihty or is the result of alternative sphcing in a sample comprising RNA from an individual being tested. The methods of S 1 nuclease analysis, RNase protection and primer extension can also be used to determine both the endpoint and the amount of a gene specific mRNA (Ausubel et al, supra). Both PCR and non-PCR based screening strategies can detect target sequences with a high level of sensitivity.

The preferred method, according to the invention, is target amphfication. According to this method, the target nucleic acid sequence is amplified with polymerases. One particularly preferred method using polymerase-driven amphfication is PCR (described above). The polymerase chain reaction and other polymerase-driven amphfication assays can achieve over a million-fold increase in copy number through the use of polymerase-driven amphfication cycles. PCR primers useful for target amphfication according to the invention, wih be designed to amplify a region of DNA containing one or more polymorphisms. Ahele specific primers (comprising one or more polymorphisms) are also useful for detecting gene sequence variations by PCR methodologies according to the invention. The absence of a particular polymorphism wih be indicated by the absence of an amphfied product when the amphfication step is caπied out in the presence of ahele specific primers. Once amphfied, the resulting nucleic acid can be sequenced and the specific sequence of the test DNA wih be compared with the wild type sequence by using the computer programs described in Section F entitled "Identification and Characterization of Polymorphisms". Alternatively, the amphfied product wih be analyzed by Southern blot assay with nucleic acid probes. Nucleic acid probes, useful according to the invention, will be specifically hybridizable to a mutant form of a gene but not to the wild type gene due to the presence of one or more polymorphisms.

When a probe comprising the target sequence, according to the invention, is used to detect the presence of the target sequences via non PCR-based strategies, (for example, in screening for osteoarthritis susceptibihty), the biological sample to be analyzed, such as blood or serum, may be treated, if deshed, to extract the nucleic acids (as described above). The sample nucleic acids (isolated from a biological sample or amphfied by PCR) may be prepared in various ways to facihtate detection of the target sequence; e.g. denaturation, restriction digestion, electrophoresis or dot blotting.

Preferably, the targeted region of the nucleic acids being analyzed are at least partiahy single-stranded to form hybrids with the targeting sequence of the probe. If the sequence is naturally single-stranded, denaturation will not be required. However, if the sequence is double-stranded, the sequence wih probably need to be denatured. Denaturation can be carried out by various techniques known in the art.

To detect the presence of a sequence variation in a gene, according to the invention, analyte nucleic acid and probe wih be incubated under conditions which promote stable hybrid formation of the target sequence in the probe with the putative targeted sequence in the sample DNA. If the region of the probe which is used to bind to the analyte is designed to be completely complementary to the targeted region, high stringency conditions are desirable in order to prevent false positives. However, conditions of high stringency wih be used only if the probes are complementary to regions of the chromosome which are unique, in the genome. The stringency of hybridization is determined by a number of factors (described above). Detection, if any, of the resulting hybrid is usuahy accomphshed by the use of labeled probes. Alternatively, the probe may be unlabeled, but may be detectable by specific binding with a hgand which is labeled, either directly or indirectly. Suitable labels, and methods for labeling probes and hgand are known in the art, and are described in Section C entitled "Production of a Nucleic Acid Probe".

^• Accordingly, the foregoing screening method may be modified to identify individuals having a gene containing a neutral polymorphism not associated with osteoarthritis, by preferably amphfying DNA fragments of a gene derived from a particular individual. The amphfied DNA fragments are sequenced and the sequence is compared to the consensus gene sequence containing neutral polymorphisms. At this time, differences between the individual's coding sequence for a gene and a consensus sequence for the same gene are determined wherein the presence of any neutral polymorphisms and the absence of a polymorphisms not previously identified as neutral polymorphisms can be correlated with an absence of increased genetic susceptibihty to osteoarthritis resulting from a mutation in a gene coding sequence.

In another embodiment of the invention, detection of a polymorphism wih be performed by detecting loss of a restriction enzyme recognition site due to the presence of one or more polymorphisms. According to this embodiment, a polymorphism wih be detected with a polynucleotide probe that is capable of detecting a restriction enzyme fragment containing the polymorphism, wherein the fragment is of a size that can be easily separated on an agarose gel and visualized by Southern blot analysis. A polynucleotide probe according to this embodiment of the invention can be specific for a sequence witliin the candidate gene or outside of the candidate gene.

It is also contemplated witliin the scope of this invention that the nucleic acid probe assays of this invention wih employ a mixtare of nucleic acid probes capable of detecting a gene. Thus, in one example to detect the presence of a gene in a test sample, more than one probe complementary to a gene is employed and in particular the number of different probes is alternatively 2, 3, or 5 different nucleic acid probe sequences. In another example, to detect the presence of mutations in the gene sequence in a patient, more than one probe complementary to a gene is employed wherein the probe mixtare includes probes capable of binding to the ahele- specific mutations identified in populations of patients with alterations in a gene. In this embodiment, any number of probes can be used, and wih preferably include probes conesponding to the major gene mutations identified as predisposing an individual to osteoarthritis.

Northern blot analysis, SI nuclease analysis, RNase protection and primer extension (Ausubel et al, supra) are also methods according to the invention for detecting changes in mRNA resulting from the presence of one or more polymorphisms in the sequence of a gene.

Additionahy, of the methods of genotyping described in Section F entitled "Identification and Characterization of Polymorphisms" can be used for diagnostics according to the invention.

2. Peptide Diagnosis and Diagnostic Kits

Osteoarthritis can also be detected on the basis of an alteration of the wild-type polypeptide. Such alterations can be determined by sequence analysis in accordance with conventional techniques. More preferably, antibodies (polyclonal or monoclonal) are used to detect differences in, or the absence of peptides derived from a gene of interest. The antibodies maybe prepared as described above in Section I entitled "Preparation of Antibodies". Preferably, antibodies wih immunoprecipitate the protein product of a gene from solution as weh as react with the protein product of a gene on Western or immunoblots of polyacrylamide gels. Antibodies useful according to the invention wih also detect the protein product of a gene in paraffin or frozen tissue sections, using immunocytochemical techniques.

Prefeπed embodiments relating to methods for detecting wild type or mutant forms of the protein product of a gene include enzyme hnked immunosorbent assays (ELISA), radioimmunoassay (RIA), immunoradiometric assays (IRMA) and immunoenzymatic assays (TEMA), including sandwich assays using monoclonal and/or polyclonal antibodies. Exemplary sandwich assays are described by David et al. In U.S. Pat. Nos. 4,376,110 and 4,486,530, hereby incorporated by reference.

3. Drug Screening

This invention is particularly useful for screening therapeutic compounds by using the mutant gene or protein product or binding fragment of the gene in any of a variety of drug screening techniques.

The protein product or fragment of a gene employed in such a test may either be free in solution, affixed to a sohd support, expressed on the surface of a ceh, or located mtracehularly. One method of drug screening utilizes eukaryotic or procaryotic host cehs which are stably transformed with a recombinant polynucleotide expressing the polypeptide or fragment, preferably in competitive binding assays. Such cehs, either in viable or fixed form, can be used for standard binding assays. In particular, these cehs can be used to measure formation of a complex comprising the protein product or fragment of a gene and the agent being tested. Alternatively, these cehs can be used to determine if the formation of a complex between the protein product or fragment of a gene and a known hgand is interfered with by an agent being tested.

Thus, the present invention discloses methods useful for drag screening wherein such methods comprise Contacting a candidate drug with a polypeptide or fragment derived from a gene and assaying (i) for the presence of a complex between the drag and the polypeptide derived or fragment derived from a gene, or (ii) for the presence of a complex between the polypeptide or fragment derived from a gene and a hgand, by methods weh known in the art. Preferably, the polypeptide or fragment derived from a gene is labeled for use in competitive binding assays. Methods for producing a labeled protein by in vitro translation are described in Section J entitled "Preparation of a Labeled Protein". Free polypeptide or fragment wih be separated from that present in a proteimprotein complex, and the amount of free (i.e., uncomplexed) label wih be used as a measure of the binding of the test drag to the polypeptide or the abihty of the test drug to interfere with protein:hgand binding.

Another method of drug screening ahows for high throughput screening for compounds exhibiting suitable binding affinity to the polypeptides and is described in detail in Geysen, WO 84/03564. According to this method, large numbers of different smah peptide test compounds are synthesized on a sohd substrate, such as plastic pins or another suitable surface. The peptide test compounds are reacted with the polypeptides or peptide fragments derived from a gene, and washed. Bound polypeptide is then detected by methods weh known in the art. Purified protein can be coated directly onto plates for use in the aforementioned drag screening techniques. Alternatively, non-neutralizing antibodies to the polypeptide can be used to capture the polypeptide or peptide fragment of interest and immobilize it on the sohd support.

Competitive drug screening assays in which neutralizing antibodies capable of specifically binding the polypeptide of interest compete with a test compound for binding to the polypeptide or fragments thereof of interest are also useful according to the invention. According to this method, antibodies can be used to detect the presence of any test peptide which shares one or more antigenic determinants with the polypeptide of interest.

An additional technique for drag screening involves the use of host eukaryotic ceh lines or cells (such as described above) which have a gene that produces a defective protein. According to this method, the host ceh lines or cehs are grown in the presence of a test drag compound. The rate of growth of the host cehs is measured to deteπnine if the compound is capable of regulating the growth of cehs expressing a nonfunctional protein product of a gene. Alternatively, the abihty of the test compound to restore the function of the mutant gene protein can be measured by using an appropriate in vitro assay for function of the protein product of a gene. Suitable in vitro functional assays are described in Section F entitled "Identification and Characterization of Polymorphisms". If the host cell lines or cehs express a protein product of a gene that exhibits an abenant pattern of cehular locahzation, the abihty of the test compound to alter the cehular locahzation of the protein wih be determined. Changes in the cehular locahzation of a protein of interest wih be detected by performing cehular fractionation studies with biosyntheticahy labeled cehs. Alternatively, the cehular locahzation of a protein of interest can be determined by immunocytochemical methods well known in the art.

A method of drag screening may involve the use of host eukaryotic ceh lines or cehs (described above) which have an altered gene that demonstrates an abenant pattern of expression. By aberrant pattern of expression is meant the level of expression is either abnormally high or low, or the temporal pattern of expression is different from that of the wild type gene. The abihty of a test drug to alter the expression of a mutant form of a gene can be measured by Northern blot analysis, S 1 nuclease analysis, primer extension or RNase protection assays. Alternatively, if a mutant form of a gene contains an polymorphisms in the promoter region of a gene, cehs can be engineered to express a reporter construct comprising a mutant gene promoter driving expression of a reporter gene (e.g. CAT, luciferase, green fluorescent protein). These cehs can be grown in the presence of a test compound and the abihty of a test compound to alter the level of activity of the mutant gene promoter can be determined by standard assays for each reporter gene which are weh known in the art. Candidate Drugs

A "candidate drag" as used herein, is any compound with a potential to modulate a phenotype associated with a particular disease according to the invention.

A candidate drag is tested in a concentration range that depends upon the molecular weight of the drug and the type of assay. For example, for inhibition of protein/protein complex formation, smah molecules (as defined below) may be tested in a concentration range of 1 pg - 100 mg/ml, preferably at about 100 pg - 10 ng/ml; large molecules, e.g., peptides, may be tested in the range of 10 ng - 100 mg/ml, preferably 100 ng - 10 mg/ml.

Candidate drug compounds from large hbraries of synthetic or nataral compounds can be screened. Numerous means are currently used for random and directed synthesis of saccharide, peptide, and nucleic acid based compounds. Synthetic compound hbraries are commerciahy available from a number of companies including Maybridge Chemical Co. (Trevihet, Cornwall, UK), Comgenex (Princeton, NJ), Brandon Associates (Merrimack, NH), and Microsource (New Mihord, CT). A rare chemical hbrary is available from Aldrich (Milwaukee, WI). Combinatorial hbraries are available and can be prepared. Alternatively, hbraries of nataral compounds in the form of bacterial, fungal, plant and animal extracts are available from e.g., Pan Laboratories (BotheU, WA) or MycoSearch (NC), or are readily produceable by methods weh known in the art. Additionally, nataral and synthetically produced hbraries and compounds are readily modified through conventional chemical, physical, and biochemical means. Useful compounds may be found within numerous chemical classes, though typically they are organic compounds, and preferably smah organic compounds. Smah organic compounds have a molecular weight of more than 50 yet less than about 2,500 daltons, preferably less than about 750 daltons, more preferably less than about 350 daltons. Exemplary classes include heterocycles, peptides, saccharides, steroids, and the like. The compounds maybe modified to enhance efficacy, stabihty, pharmaceutical compatibihty, and the like. Structural identification of an agent may be used to identify, generate, or screen additional agents. For example, where peptide agents are identified, they may be modified in a variety of ways to enhance their stabihty, such as using an unnatural a ino acid, such as a D-amino acid, particularly D-alanine, by functionalizing the amino or carboxylic terminus, e.g. for the amino group, acylation or alkylation, and for the carboxyl group, esterification or amidification, or the like.

Determination of Activity of a Drug

A candidate drag, assayed according to the invention as described above, is determined to be effective if its use results in a change of about 10% of a phenotype associated with a disease according to the invention.

The level of modulation by a candidate modulator of a phenotype associated with a disease according to the invention, maybe quantified using any acceptable limits, for example, via the fohowing formula, which describes detections performed with a radioactively labeled probe (e.g., a radiolabeled antibody in an immunobinding experiment or a radiolabeled nucleic acid probe in a Northern hybridization).

(CPM_Control - CPM_Sample) Percent Modulation = xlOO

(CPM_Control)

where CPM_Control is the average of the cpm in antibody/hgand complexes or on Northern blots resulting from assays that lack the candidate modulator (in other words, untreated controls), and CPM_Sarople is the cpm in antibody/hgand complexes or on Northern blots resulting from assays containing the candidate modulator. A similar calculation is performed where the assay comprises use of a labeling system or system of measuring enzymatic activity in which there is a linear relationship between the amount of label detected and the amount of protein or nucleic acid being represented per unit of label or the amount of protein or nucleic acid represented by a unit of enzymatic activity.

4. Rational Drug Design

Rational drag design is useful for producing either structural analogs of biologically active polypeptides of interest or smah molecules with which polypeptides of interest interact (e.g., agonists, antagonists, inhibitors) in order to design drags which are, for example, more active or stable forms of the polypeptide, or which enhance or interfere with the function of a polypeptide in vivo. See, e.g., Hodgson, 1991, BioTechnology, 9:19. According to one method of rational drag design, the three- dimensional structure of a protein of interest (e.g., the polypeptide product of the gene) or, or the complex comprising the protein product of a gene in association with its hgand, is deteimined by x-ray crystallography, by computer modeling or most typicahy, by a combination of approaches. Alternatively, useful information regarding the structure of a polypeptide may be obtained by modeling based on the stractare of homologous proteins. Rational drag design has been used successfully in the development of HTV protease inhibitors (Erickson et al, 1990, Science, 249: 527).

Rational drug design may also involve the analysis of peptides derived from the protein product of a gene by an alanine scan (Wehs, 1991, Methods in Enzymol, 202: 390). According to this method, each of the amino acid residues of the peptide is sequentiahy replaced by alanine, and the effect of this amino acid substitution on the peptide' s activity is determined. This technique can be used to determine the functionally relevant regions of the peptide. Another experimental approach to rational drug design wih involve the isolation of a target- specific antibody (selected by a functional assay) and the determination of the crystal stractare of this antibody. Theoretically, this approach wih yield a pharmacore upon which subsequent drag design can be based. Alternatively, if anti-idiotypic antibodies (anti-ids) specific for a functional, pharmacologically active antibody are generated, there is no need to determine the crystallographic structure of the target-specific antibody. It is expected that the binding site of the anti-ids wih be an analog of the original receptor. The anti-id could then be used to identify and isolate potentiahy therapeutic peptides from banks of chemically or biologically produced banks of peptides. These selected peptides would then function as pharmacores.

According to these methods it may be possible to design drugs which demonstrate increased activity or stabihty of the protein product of a gene or which function as inhibitors, agonists, antagonists, etc. of the activity of a protein product of a gene. The availability of cloned gene sequences, including polymorphisms, ensures that sufficient amounts of the polypeptide product of a gene are available to facihtate analytical studies such as x-ray crystahography. Furthermore, the knowledge of the sequence of the protein product of a gene provided herein wih guide those using computer modeling techniques in place of, or in addition to x-ray crystahography.

5. Gene Therapy

The present invention also provides a method of supplying wild-type gene function to a ceh which carries a mutant ahele of a gene. By replacing a mutant gene with a wild type gene, it may be possible to reverse the symptoms of osteoarthritis in the recipient cehs. a full length version of the wild-type gene, or a fragment of the gene, may be introduced into the ceh in a vector such that the gene remains extrachromosomal and is expressed by the ceh from the extrachromosomal location. More preferably, fohowing introduction into the mutant ceh, the wild-type gene or gene fragment should recombine with the endogenous mutant gene X already present in the ceh. Such recombination requhes a double recombination event which results in the conection of the gene mutation. Vectors for introduction of genes both for recombination and for extrachromosomal maintenance are known in the art, and any suitable vector may be used. Methods for introducing DNA into cehs such as electroporation, calcium phosphate coprecipitation and lipofection are known in the art (described above). Cehs transformed with the wild-type gene can be used as model systems to study changes in the intensity of symptoms associated with osteoarthritis and drug treatments which promote such changes.

As generally discussed above, a gene or a fragment thereof, where apphcable, may be used in gene therapy methods in order to increase the amount of the expression products of such genes in cehs of patients with osteoarthritis. It may also be useful to increase the level of expression of a gene even in those cehs in which the mutant gene is expressed at a "normal" level, but the gene product is not fully functional.

It other embodiments of the invention it may be useful to increase the amount of the expression products of a mutant form of a gene in a ceh that expresses the wild type protein. Gene therapy can be carried out according to generahy accepted methods, for example, as described by Friedman, 1991, In Therapy for Genetic Diseases; T. Friedman ed., Oxford University Press, pp. 105- 121). Initiahy, the appropriate cehs from a patient with osteoarthritis would be analyzed by the diagnostic methods described above, to determine the level of production of a polypeptide from a gene and the activity of a polypeptide product of a gene. A virus or plasmid vector (see further details below), comprising a copy of a gene and suitable expression control elements, and capable of rephcating inside the cehs, wih be prepared. Suitable vectors are known and are disclosed in U.S. Pat. No. 5,252,479 and PCT published apphcation WO 93/07282. The vector wih be injected into the patient, either locahy at an appropriate site according to the invention or systemically. Gene transfer systems known in the art may be useful in the practice of the gene therapy methods of the present invention. These include viral and nonviral transfer methods, a number of viruses have been used as gene transfer vectors, including papovavirases, e.g., 5V40 (Madzak et al, 1992, J Gen Vhol, 73:1533), adenovirus (Berkner, 1992, Cun. Top. Microbiol. Immunol, 158:39; Berkner et al, 1988, BioTechniques, 6:616; Gorzigha and Kapfldan, 1992, J Vhol, 66:4407; Quantin et al, 1992, Proc. Natl. Acad. Sci. USA, 89:2581; Rosenfeld et al, 1992, Ceh, 68:143 ; Wilkinson et al, 1992, Nucleic Acids Res. 20:2233; Stratford-Peπicaudet et al, 1990, Hum. Gene Ther., 1:241), vaccinia virus (Moss, 1992, Curr. Top. Microbiol. Immunol, 158:25) adeno-associated virus (Muzyczka, 1992, Cun. Top. Microbiol. Immunol, 158:97; Ohi et al, 1990, Gene, 89:279), herpesviruses including HSV and EBV (Margolskee, 1992, Cun. Top. Microbiol. Immunol, 158:67, Johnson et al, 1992, J. Vhol, 66:2952; Fink et al, 1992, Hum. Gene Ther., 3:11; Breakfield and Geher, 1987, Mol. Neurobiol, 1:337; Freese et al, 1990, Biochem. Pharmacol, 40: 2189), and retroviruses of avian (Brandyopadhyay and Temin, 1984, Mol. Ceh. Biol, 4:749; Petropoulos et al, 1992, J. Vhol, 66:3391), marine (Miller, 1992, Cun. Top. Microbiol. Immunol, 158:1; Miher et al, 1985, Mol. Ceh. Biol, 5:431; Sorge et al, 1984, Mol. Ceh. Biol, 4:1730; Mann and Baltimore, 1985, J. Vhol, 54:401;

Miher et al, 1988, J. Virol, 62:4337), and human origin (Shimada et al, 1991, J. Clin. Invest, 88:1043);

Helseith et al, 1990, J. Virol, 64:24 16; Page et al, 1990, J. Vhol, 64: 5370; Buchschacher and

Panganiban, 1992, J. Virol, 66:2731). Most human gene therapy protocols have been based on disabled murine retiOvirases.

Nonviral gene transfer methods known in the art include chemical techniques such as calcium phosphate coprecipitation (Graham and van der Eb, 1973, Virology, 52:456; Pel cer et al, 1980,

Science, 209:1414); mechanical techniques, for example microinjection (Anderson et al, 1980, Proc.

Natl. Acad. Sci. USA, 77: 5399; Gordon et al, 1980, Proc. Natl. Acad. Sci. USA, 77: 7380; Brinster et al, 1981, Ceh, 27:223; Constantini and Lacy, 1981, Natare, 294:92); membrane fusion-mediated transfer via hposomes (Feigner et al, 1987, Proc. Natl Acad. Sci. USA, 84:7413; Wang and Huang,

1989, Biochemistry, 28:9508; Kaneda et al. 1989, J. Biol. Chem., 264:12126; Stewart et al., 1992, Hum. Gen. Ther., 3:267; Nabel et al, 1990, Science, 249:1285; Lim et al, 1992, Circulation, 83:2007); and direct DNA uptake and receptor-mediated DNA transfer (Wolff et al, 1990, Science, 247:1465; Wu et al, 1991, J. Biol. Chem., 266:14338; Zenke et al, 1990, Proc. Natl Acad. Sci. USA, 87:3655; Wu et al, 1989b, J. Biol. Chem., 264:16985; Wolff et al, 1991, BioTechniques, 11:474; Wagner et al,

1990, Proc. Natl Acad. ScLUSA, 87:3410; Wagner et al, 1991, Proc. Natl Acad SciUSA, 88:4255; Gotten et al, 1990, Proc. Natl. Acad. SciUSA, 87:4033; Curiel et al, 1991a, Proc. Natl Acad. SciUSA, 88:8850; Curiel et al, 1991b, Hum. Gene Ther., 3:147. In an approach which combines biological and physical gene transfer methods, plasmid DNA of any size is combined with a polylysine-conjugated antibody specific to the adenovirus hexon protein, and the resulting complex is bound to an adenovirus vector. The trimolecular complex is then used to infect cehs. The adenovirus vector permits efficient binding, internahzation, and degradation of the endosome before the coupled DNA is damaged. Liposome DNA complexes have been shown to be capable of mediating direct in vivo gene transfer. While in standard hposome preparations the gene transfer process is nonspecific, locahzed in vivo uptake and expression have been reported in tumor deposits, for example, fohowing direct in situ administration (Nabel, 1992, Hum. Gen. Ther., 3:399).

Gene transfer techniques which target DNA directly to an appropriate tissue, e.g., a tissue that normahy expresses the protein product of the candidate gene of the invention, is prefeπed.

Receptor-mediated gene transfer, for example, is accomphshed by the conjugation of DNA (usuahy in the form of covalently closed supercoiled plasmid) to a protein hgand via polylysine. Ligands are chosen on the basis of the presence of the conesponding hgand receptors on the ceh surface of the target cell/tissue type. These hgand-DNA conjugates can be injected directly into the blood if deshed and are directed to the target tissue where receptor binding and internahzation of the DNA-protein complex occurs. To overcome the problem of intracellular destruction of DNA, coinfection with adenovirus can be included to disrupt endosome function.

6. Peptide Therapy

Peptides which have gene activity can be supphed to cehs which carry mutant or missing aheles of a gene. Alternatively, peptides specific for a mutant form of the protein product of a gene can be supphed to cehs carrying a wild type protein. The protein product of a gene can be produced by expression of the cDNA sequence in bacteria, for example, using known expression vectors (as described in Sbction H entitled "Production of a Mutant Protein"). Alternatively, the protein product of a gene can be extracted from mammalian cehs engineered to produce the protein product of a gene of interest. In addition, the techniques of synthetic chemistry can be employed to synthesize the protein product of a gene. Any of the above techniques can provide a preparation of protein product of a gene that is substantiahy free of other human proteins. This is most readily accomphshed by caπying out protein synthesis in a microorganism or in vitro.

Active gene molecules can be introduced into cehs by microinjection or by the use of hposomes, for example. Alternatively, some active molecules may be taken up by cehs, actively or by diffusion. Extracellular apphcation of the protein product of a gene may be sufficient to decrease or reverse the physiological effects of osteoarthritis. Other molecules with the activity of a protein product of a gene (for example, peptides, drugs or organic compounds) may also be used to effect such a reversal. Modified polypeptides having substantiahy similar function may also be useful for peptide therapy.

7. Transformed Hosts

Cehs and animals which cany a mutant ahele of a gene can be used as model systems to study and test for substances which have potential as therapeutic agents. Fohowing apphcation of a test substance to the cehs, the phenotype of the ceh wih be determined. Any variety of phenotypic changes associated with osteoarthritis can be assessed, including insulin resistance and combined insulin resistance/insulin secretion detect. Assays for each of these traits are known in the art.

Animals useful for testing therapeutic agents can be selected after mutagenesis of whole animals or after treatment of germline cehs or zygotes. Such treatments include insertion of mutant aheles of a gene, usuahy from a second animal species, as weh as insertion of disrupted homologous genes. Alternatively, the endogenous gene of the animals maybe disrupted by insertion or deletion mutation or other genetic alterations using conventional techniques (Capecchi, 1989, Science, 244:1288; Valancius and Smithies, 1991, Mol Cell. Biol, 11:1402; Hasty et al, 1991, Natare, 350:243; Sbinkai et al, 1992, Cell, 68:855; Mombaerts et al, 1992, Ceh, 68:869; Philpott et al, 1992, Science, 256:1448; Snouwaert et al, 1992, Science, 257:1083; Donehower et al, 1992, Natare, 356;215). Fohowing the administration of test substances, the physiological changes associated with osteoarthritis wih be assessed. If the test substance prevents or suppresses any of these physiological changes, then the test substance wih be considered a candidate therapeutic agent for the treatment of osteoarthritis. These animal models provide an extremely important testing vehicle for potential therapeutic products.

8. Use of a Polynucleotide as a Unique Sequence Marker:

Polynucleotides can be used to mark objects or substances for the purposes of later identification. Thus, polynucleotides of the invention are useful for tracking the manufacture and distribution of a large number of diverse substances, including but not limited to: (1) nataral resources such as animals, plants, oil, minerals, and water; (2) chemicals such as drags, solvents, petroleum products, and explosives; (3) commercial by-products including pollutants such as radioactive or other hazardous waste; and (4) articles of manufacture such as guns, typewriters, automobiles and automobile parts. A nucleic acid according to the invention, when used as a marker, thus aids in the determination of product identity and so provides information useful to manufacturers and consumers. Polynucleotides have the advantage over other marking materials of being readily amplifiable through the use of polymerase chain reaction (PCR) technology. The method of PCR is weh known in the art. PCR is performed as described by Mulhs & Faloona, 1987, Methods Enzymol, 155:335, herein incorporated by reference. It is the unique sequence of a polynucleotide which renders it useful as a marker, since thesequence, or a characteristic pattern derived from its sequence, confers a property on the polynucleotide which permits it to be tracked.

It is contemplated that a novel polynucleotide sequence of the invention, or fragments or derivatives of it may be used as markers by their attachment to or mixtare in objects or substances to be marked. Methods for marking various classes of substances and later detection of the tags in those substances are disclosed in U.S. Patent Nos. 5,451,505, and 5,643,728. Briefly, the use of a polynucleotide of the invention as a marker may entail combining a polynucleotide with the substance or object to be marked, using methods appropriate to that substance or object; and detecting the marker through amphfication of the polynucleotide sequence using PCR technology, fohowed by either sequence analysis or identification by other means known in the art (e.g., hybridization assays).

The methods of applying a marker nucleic acid to a substance or object and subsequent detection of that nucleic acid wih vary depending upon the natare of the substance or object and the environment to which it wih be exposed. For example, inert solids such as paper, many pharmaceutical products, wood, some foodstuffs, etc., can be either processed with the marker nucleic acid, or the nucleic acid may be sprayed onto their surfaces. Chemically active substances, such as foodstuffs with enzymatic activity, polymers with charged groups, or acidic pharmaceuticals may require that a protective composition (e.g., hposomes) be added to the nucleic acid being used as a marker.

In order to mark liquids, the nucleic acid may be mixed directly with the hquid, or, if the chemical natare of the hquid is not compatible with this approach (i.e. , nucleic acids are not soluble in the hquid), the nucleic acid maybe mixed with a detergent to enhance its solubihty. Containerized gases may be marked simply by adding a nucleic acid to the container in dry form, as it wih be dispersed throughout the gas as the gas is released.

The amount of nucleic acid to add to a substance as a marker wih also vary with the given situation, as wih the detection strategy. PCR technology, however, ahows the amplification and detection of as little as one molecule from a sample. Other means of detection, such as hybridization assays requhe that more nucleic acid be recovered from a sample to efficiently detect it. PCR can be combined with a hybridization assay, however, to enhance the sensitivity of the method.

A nucleic acid sequence used as a marker wih generahy be from 20 to 1,000 bases long, and preferably wih be 60 to 1,000 bases long when PCR is to be used to detect the marker.

One example of a substance for which nucleic acid marking is suited is gunpowder. Marked gunpowder may be prepared as fohows: 1) add 16 ng of nucleic acid bearing the chosen marker sequence (derived from a polynucleotide of the invention) to 1 ml of distihed water; 2) mix the solution of nucleic acid with 1 g of nitrocellulose-based gunpowder; and 3) dry in ah or under vacuum at 85°C. To recover the marker from gunpowder: 1) wash the gunpowder sample with 1 ml of distihed water; 2) add 50 ml of the wash solution to a standard PCR mix, or, alternatively, place gunpowder flakes directly into a 100 ml PCR mix; and 3) amplify according to standard PCR methods using primers which anneal at opposite ends and on opposite strands of the sequence used as a marker (anneahng and extension conditions wih depend upon the exact sequences chosen for oligonucletide primers, and may be adjusted according to methods known in the art).

Another example of a substance which may be marked with a nucleic acid according to the invention is ink. To prepare marked ink sample: 1) if the ink is water insoluble, mix the nucleic acid with detergents as for oil. If the ink is water soluble, add nucleic acid directly to the ink to a concentration of about 1 to 20 ng per ml. To recover the marker from ink, proceed as for oils and medicines.

In the above examples, the presence of an amphfication product of the proper size (visuahzed, for example by gel electrophoresis alongside nucleic acid size markers followed by ethidium bromide staining of the gel, according to standard methods) wih indicate the presence of the marker in the sample. In some instances, the PCR product may be further subjected to hybridization analysis or to sequencing to enhance the accuracy of the method. A method of hybridization analysis which can be used is described herein.

9. Use of a Polynucleotide of the Invention as a Marker for Chromosome Mapping:

Because a polynucleotide of the invention is novel, (that is, its sequence is unique),it is useful as a marker for cliromosomal mapping. There are a number of methods of chromosomal mapping known in the art. Prominent among them is the variant of the in situ hybridization technique known as "Fluorescence In Situ Hybridization", or FISH. Details of methods and solutions used for in situ hybridization are weh-known in hie art. There are many variations of the FISH technique itself, however the basic approach is similar in each case. Essentially, in situ hybridization of cehs, nuclei, or metaphase chromosome spreads is performed with a polynucleotide probe either directly labeled with' a fluorochrome, or labeled with a moiety which wih be bound by a fluorochrome tagged entity. The hybridized probe is visuahzed by inadiation of the sample with hght in the wavelength which excites fluorescence from the fluorochrome. When combined with standard methods of karyotyping known in the art, this method ahows the polynucleotide sequence to be locahzed to a particular arm of a particular chromosome. Once mapped to a specific chromosome, the location of the novel polynucleotide sequence on that chromosome maybe further locahzed by in situ hybridization along with probes specific for known genes or sequences, labeled with other fluorescent tags which ahow the differentiation of the signals from the different probes. Such an approach and various adaptations of it ahows the locahzation of the novel gene relative to a known gene. Methods of generating and using fluorescence-labeled polynucleotide probes for FISH and chromosome mapping are known in the art (for example, see Malcolm et al, 1981, Ann. Hum. Genet, 45:134; Bar-Am et al, 1992, Genes. Chromosomes & Cancer, 4:314; Pinkel et al, 1988, Proc. Natl. Acad. Sci. USA, 85:9138; U.S. Patent No. 5,728,527). Additional variations of the chromosome mapping method utihze a PCR approach (Dionne et aL, 1990, BioTechniques, 8(2):190 and Iggo et al, 1989, Proc. Natl. Acad. Sci. USA, 86:6211).

In addition to being able to determine the chromosomal location of the novel polynucleotide, similar technology, in which FISH is combined with flow cytometry, wih ahow the polynucleotide of the invention to be used to sort chromosomes, nuclei, or whole cehs containing various dosages (i.e., gene copy numbers) of the gene encoding that polynucleotide (Hulfdin et al, 1998, Nuc. Acids Res., 26:3651). The novel polypeptide may also be useful as a diagnostic indicator of a disease, including but not limited to tliose hsted in Table I (Kuo et al, 1990, Am. J. Hum. Genet, 47:A119).

10. Use of a Polynucleotide of the Invention as a Marker for Analysis of Forensic

Materials Forensic science depends heavily on methods for deteririining the source of various compounds associated with criminal activity. In particular, the identification of individuals involved in criminal activity through analysis of substances found at the crime scenes is critical. Such identification is possible with genetic typing, which involves the determination of the genotype of an individual with regard to loci which are polymorphic within the population. As used herein, "polymorphic" refers to a gene or other segment of DNA which shows nucleotide sequence variability from individual to individual. The use of PCR techniques and nucleotide probes to detect even single nucleotide changes in a polynucleotide sequence has revolutionized the field of forensic serology (see Reynolds and Sensabaugh, 1991, Anal. Chem., 63:2). For an example of polymorphisms useful for forensic identification and methods of typing samples with regard to those polymorphisms, see U.S. Patent # 5,273,883. If a polynucleotide of the invention is found to have nucleotide sequence variation among individuals within a population, it may be useful in the analysis of forensic samples. There are a number of methods known to those skihed in the art for typing nucleic acids with regard to polymorphisms. It should be understood that any such method is acceptable according to the invention. One particular method is termed the "reverse dot blot" method. The basic steps involved are: 1) ohgonucleotides bearing the sequences of various polymorphic forms of the polynucleotide region to be analyzed are bound to membranes; 2) labeled, PCR-amphfied fragments, derived from the sample to be genotyped, and conesponding to the polymorphic region ("target DNA") are ahowed to hybridize to the bound ohgonucleotides under conditions which only ahow the hybridization of molecules with 100% complementary sequences; 3) unbound target DNA is removed; and 4) hybridized molecules are detected.

The specific genotype of the individual from whom the target sample was obtained (amphfied), with regard to the polymorphic region of a polynucleotide of the invention, may thus be determined by screening a panel of probes containing the known polymorphic sequence variations of that region. It should be understood that the hybridization conditions may be adjusted by one of skill in the art so that limited amounts of non-complementarity, including single base mismatches, may be detected with this method.

Q. Pharmaceutical Compositions-Prevention and Treatment

1. Administration of Pharmaceutical Compositions

Administration of pharmaceutical compositions is accomphshed orahy or parenterally. Methods of parenteral dehvery include topical, intra-arterial (directly to the tumor), intramuscular, subcutaneous, intrameduhary, mtrathecal, intraventricular, intravenous, intraperitoneal, or intranasal administration. In addition to the active ingredients, these pharmaceutical compositions may contain suitable pharmaceutically acceptable caπier preparations which can be used pharmaceutically.

Pharmaceutical compositions for oral administration can be formulated using pharmaceutically acceptable carriers weh known in the art in dosages suitable for oral administration. Such carriers enable the pharmaceutical compositions to be formulated as tablets, pihs, dragees, capsules, hquids, gels, syrups, sluπies, suspensions and the like, for ingestion by the patient.

Pharmaceutical preparations for oral use can be obtained through combination of active compounds with sohd excipient, optionahy grinding a resulting mixtare, and processing the mixture of granules, after adding suitable auxiliaries, if deshed, to obtain tablets or dragee cores. Suitable excipients are carbohydrate or protein fihers such as sugars, including lactose, sucrose, mannitol, or sorbitol; starch from corn, wheat, rice, potato, or other plants; cehulose such as methyl cehulose, hydroxypropylmethyl-cehulose, or sodium carboxymethyl cehulose; and gums including arabic and tragacanth; and proteins such as gelatin and cohagen. If deshed, disintegrating or solubilizing agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, alginic acid, or a salt thereof, such as sodium alginate.

Dragee cores are provided with suitable coatings such as concentrated sugar solutions, which may also contain gum arabic, talc, polyvinylpynohdone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. Dyestaffs or pigments maybe added to the tablets or dragee coatings for product identification or to characterize the quantity of active compound, ie, dosage.

Pharmaceutical preparations which can be used orahy include push-fit capsules made of gelatin, as weh as soft, sealed capsules made of gelatin and a coating such as glycerol or sorbitol. Push-fit capsules can contain active ingredients mixed with a filler or binders such as lactose or starches, lubricants such as talc or magnesium stearate, and, optionahy, stabilizers. In soft capsules, the active compounds may be dissolved or suspended in suitable hquids, such as fatty oils, hquid paraffin, or hquid polyethylene glycol with or without stabihzers.

Pharmaceutical formulations for parenteral administration include aqueous solutions of active compounds. For injection, the pharmaceutical compositions of the invention may be formulated in aqueous solutions, preferably in physiologicahy compatible buffers such as Hank's solution, Ringer' solution, or physiologicahy buffered saline. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cehulose, sorbitol, or dextran. Additionally, suspensions of the active solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or hposomes. Optionahy, the suspension may also contain suitable stabihzers or agents which increase the solubihty of the compounds to ahow for the preparation of highly concentrated solutions.

For topical or nasal administration, penetrants appropriate to the particular barrier to be permeated or used in the formulation. Such penetrants are generally known in the art.

2. Manufacture and Storage

The pharmaceutical compositions of the present invention may be manufactured in a manner that known in the art, e.g. by means of conventional mixing, dissolving, granulating, dragee-making, levitating, emulsifying, encapsulating, entrapping or lyophihzing processes. The pharmaceutical composition may be provided as a salt and can be formed with many acids, including but not limited to hydrochloric, sulfuric, acetic, lactic, tartaric, malic, succinic, etc... Salts tend to be more soluble in aqueous or other protonic solvents that are the conesponding free base forms. In other cases, the preferred preparation maybe a lyophilized powder in lmM-50 mM histidine, 0.1%-2% sucrose, 2%-7% mannitol at a PhRange of 4.5 to 5.5 that is combined with buffer prior to use.

After pharmaceutical compositions comprising a compound of the invention formulated in a acceptable caπier have been prepared, they can be placed in an appropriate container and labeled for treatment of an indicated condition with information including amount, frequency and method of administration.

3. Therapeutically Effective Dose

Pharmaceutical compositions suitable for use in the present invention include compositions wherein the active ingredients are contained in an effective amount to achieve the intended purpose. The determination of an effective dose is weh within the capabihty of those skihed in the art.

For any compound, the therapeuticahy effective dose can be estimated initiahy either in ceh culture assays, or in animal models, usuahy mice, rabbits, dogs, or pigs. The animal model is also used to achieve a desirable concentration range and route of administration. Such information can then be use to determine useful doses and routes for administration in humans.

A therapeuticahy effective dose refers to that amount of protein or its antibodies, antagonists, or inhibitors which ameliorate the symptoms or conditions. Therapeutic efficacy and toxicity of such compounds can be determined by standard pharmaceutical procedures in ceh cultures or experimental animals, eg, ED50 (the dose therapeuticahy effective in 50% of the population) and LD50 (the dose lethal to 50% of the population). The dose ratio between therapeutic and toxic effects is the therapeutic index, and it can be expressed as the ratio, LD50/ED50. Pharmaceutical compositions which exhibit large therapeutic indices are preferred. The data obtained from ceh culture assays and animals studies is used in formulating a range of dosage for human use. The dosage of such compounds hes preferably within a range of circulating concentrations that include the ED50 with httle or no toxicity. The dosage varies within this range depending upon the dosage from employed, sensitivity of the patient, and the route of administration.

The exact dosage is chosen by the individual physician in view of the patient to be treated. Dosage and administration are adjusted to provide sufficient levels of the active moiety or to maintain the deshed effect. Additional factors which may be taken into account include the severity of the disease state; age, weight and gender of the patient; diet, time and frequency of administration, drag combinations), reaction sensitivities, and tolerance/response to therapy. Long acting pharmaceutical compositions might be administered every 3 to 4 days, every week, or once every two weeks depending on a hah-hfe and clearance rate of the particular formulation.

Dosage amounts may vary from 0.1 to 100,000 micrograms per person per day, for example, lug, lOug, lOOug, 500 ug, lmg, lOmg, and even up to a total dose of about lg per person per day, depending upon the route of administration. Guidance as to particular dosages and methods of dehvery is provided in the literature. See U.S. Patent Nos. 4,657,760; 5,206,344; or 5,225,212, hereby incorporated by reference. Those skihed in the art wih employ different formulations for nucleotides than for proteins or their inhibitors. Similarly, dehvery of polynucleotide or polypeptides wih be specific to particular cehs, conditions, locations, etc...

Without further elaboration, it is beheved that one skihed in the art can, using the preceding description, utihze the present invention to its fullest extent. The fohowing embodiments are, therefore, to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever.

The disclosures of ah patents, apphcations, and publications mentioned above and below, including U.S. Ser. No. 60/342,603, are hereby expressly incorporated by reference.

EXAMPLES

1. Establishment of an Association Between a Given Polynucleotide Sequence and Diabetes

A polynucleotide sequence according to the invention containing a mutation which is beheved to be associated with a disease, can be statisticahy linked to that disease by hnkage analysis. An animal model system exhibiting a particular phenotypic defect that is characteristic of the disease of interest is selected. A series of genetic crosses is performed in this animal model system between individuals having an observable mutant phenotype and normal individuals of a control strain. At least one disease-related locus or a chromosomal marker that does not comprise a disease related locus is used as a marker in these crosses. If a statisticahy significant pattern of non-random assortment of the mutant trait with a marker locus is observed, the trait is linked to the marker locus.

Similarly, linkage analysis can be performed on an existing human or other mammalian pedigree. According to this method, numerous genetic loci from affected and unaffected family members are compared. Non-random assortment of a given genetic marker between affected and unaffected family members relative to the distributions observed for other genetic loci indicates that the marker (for example, a variant isoform of a gene) either contributes to the disease or is in physical proximity to another that does so.

If either approach demonstrates a non-random assortment of the disease-related phenotype with a marker locus, this is indicative of an association between the gene underlying the defect and that locus. Because the strength of any conclusion drawn from linkage analysis is statisticahy-based, the accuracy of the results is thought to be proportional to the number of crosses or family members and genetic loci analyzed.

2. Screening Assay For a Disease A polynucleotide sequence according to the invention can be used as a marker for a normal phenotype or for a phenotype associated with a disease of interest.

If it can be demonstrated by the methods of phenotyping, described above, that a particular sequence is associated with a disease phenotype, this sequence can be used as a marker for a particular disease. A sequence of interest can be used as a probe to screen genomic DNA from individuals by Southern blot analysis according to the method described above. If the sequence of interest is detected by Southern blot analysis, and the presence of this sequence is confirmed by direct sequencing, it can be concluded that the individual from which the genomic DNA has been isolated has an increased frequency for the development of the disease for which the sequence is a marker. The marker can also be used as a disease indicator according to the method of PCR. A genomic DNA sample of interest can be analyzed in a PCR reaction wherein one of the primers contains the marker sequence. If the marker sequence is present in the sample DNA, a PCR product wih be produced. Alternatively, the PCR primers can be designed such that they amplify a region containing the marker sequence. The amphfied product can be analyzed by hybridization methods, described above, to determine the presence of the sequence of interest.

3. Use of a Given Polynucleotide as a Target for Drug Screening

A polynucleotide according to the invention, containing a mutation which is beheved to be associated with a disease can be used a target for drug screening.

One method of drag screening utihzes eukaryotic or procaryotic host cehs which are stably transformed with a polynucleotide according to the invention and either exhibit a particular phenotype characteristic of the presence of the polynucleotide or express a polypeptide or fragment encoded by the polynucleotide. Such cehs, either in viable or fixed form, can be used for standard competitive binding assays. In particular, these cehs can be used to measure formation of a complex comprising the protein product or fragment of a polynucleotide according to the invention and the agent being tested. Alternatively, these cehs can be used to determine if the formation of a complex between the protein product or fragment of a polynucleotide according to the invention and a known hgand is interfered with by an agent being tested. An alternative method for drug screening involves using of eukaryotic ceh lines or cehs (such as described above) which contain a polynucleotide according to the invention that produces a defective protein. According to this method, the host ceh lines or cehs are grown in the presence of a test drug. The rate of growth of the host cehs is measured to determine if the compound is capable of regulating the growth of cehs expressing a nonfunctional protein product of the polynucleotide according to the invention. Preferably, a drag that is useful according to the invention wih increase or decrease the growth rate of a ceh by at least 10%. Alternatively, the abihty of the test compound to restore the function of the mutant gene protein by at least 10% can be measured by using an appropriate in vitro assay for function of the protein product of a gene (as described in Section F entitled "Identification and Characterization of Polymorphisms"). If the host ceh lines or cehs express a protein product of a gene that exhibits an aberrant pattern of cehular locahzation, the abihty of the test compound to alter the cehular locahzation of the protein by at least 10% will be determined. Changes in the cehular locahzation of a protein of interest wih be detected by performing cehular fractionation studies with biosyntheticahy labeled cehs. Alternatively, the cehular locahzation of a protein of interest can be determined by immunocytochemical methods weh known in the art.

A method of drug screening may also involve the use of host eukaryotic ceh lines or cehs (described above) which have an altered gene that demonstrates an aberrant pattern of expression. By abeneant pattern of expression is meant the level of expression is either abnormahy high or low, or the temporal pattern of expression is different from that of the wild type gene. The abihty of a test drag to alter the expression of a mutant form of a gene by at least 10% can be measured by Northern blot analysis, SI nuclease analysis, primer extension or Rnase protection assays, as described above. Alternatively, if a mutant form of a gene contains a polymorphism in the promoter region of a gene, cehs can be engineered to express a reporter construct comprising a mutant gene promoter driving expression of a reporter gene (e.g. CAT, luciferase, green fluorescent protein). These cehs can be grown in the presence of a test compound and the abihty of a test compound to alter the level of activity of the mutant gene promoter can be determined by standard assays for each reporter gene which, are weh known in the art.

A transgenic animal whose genomic DNA contains a polynucleotide associated with a particular phenotypic defect that is characteristic of the disease of interest, and a normal, control anomal (not containing the polynucleotide) can be treated with a candidate drag according to the invention. The abihty of a candidate drug to ameliorate symptoms of the disease, by at least 10%, wih be analyzed by assessing the disease syptoms and their amelioration.

4. Selection of Osteoarthritis Candidate Gene Set

Genes involved in osteoarthritis

Key pathogenic processes involved in osteoarthritis are:

1. chondrocyte differentiation, development, apoptosis and signalling

2. cartilage components and synthesis : proteoglycans, hyaluronan synthases, extracehular matrix molecules 3. cartilage degradation: cathepsin proteases and matrix metahoproteinases, their inhibitors

4. bone remodelling signals (e.g. RANK/RANKL): BMPs, TGFbeta, interleukins, their receptors and antagonists, downstream signaling.

5. synovial fluid components 6. systemic factors influencing bone and cartilage remodelling: leptin, estrogen, progesterone, inflammatory cytokines, retinoic acid

Polymorphisms at the fohowing genes have been reported in the literature to be involved with increased risk of osteoarthritis. They include components of the extracehular matrix, and bone- remodelling signalling components (Table 2)

With the aim of expanding and improving on the current limited knowledge of osteoarthritis genetic predisposition, we have cohected over 500 candidate bone and cartilage remodelling genes using the fohowing methods:

1. extensive literature search for genes involved in relevant biochemical pathways and physiological processes

2. analysis and comparisons of cDNA hbraries witliin the Incyte Lifeseq® database from relevant normal and diseased tissues and in vitro modelling systems

3. co-expression analysis using Incyte's "Guilt by Association" algorithm which identifies novel genes in key biochemical pathways by comparing the expression patterns of genes within the Lifeseq® database

5. Polymorphisms in Genes Associated with Osteoarthritis

The osteoartbritis candidate gene hst was compiled using gene or gene sequences selected from literature sources, using sequence homology, hbrary subtraction and expression analysis.

Expression analysis was performed using "quilt-by-association" queries to identify Incyte- novel and known genes not previously associated with diabetes which have similiar expression patterns to genes known to be involved in diabetes or related conditions. Guilt-by-association analysis was performed as described in Walker et al. 1999 Genome Res 9:1198; Walker et al. 1999 Ismb :282; and US Patent Apphcation 09/226,994 entitled "Insulin-Synthesis Genes" (Atty Docket No: PB-0008 US) filed January 7, 1999, ah of which are incorporated by reference.

Polymorphism discovery was by fSSCP as decribed in section F "Identication and Characterization of Polymorphisms", subsection b5 for polymorphisms referred to in Table 3 for source wetSNPs. Polymorphisms referred to as source isSNPs were discovered as described in section F "Identification and Characterization of Polymorphisms", subsection a. Polymorphisms refened to as source dbSNPs are polymorphisms in pubhc genomic sequence where gene stractare is unknown. The polymorphisms were mapped to cDNA sequences in the LifeSeqGold database (Incyte) to identify gene identity.

6. Frequency of Polymorphisms in Diabetes Associated Genes and Polynucleotides in

Various Populations

Polymorphisms identified in EXAMPLES 4 and 5 were genotyped against populations described below by fSSCP or FP-TDI as described above. The results of the population frquency studies are given in Table 2. Two panels of human DNA have been developed to support the identification of frequent

SNPs within an ethnically diverse population. The genomic Human Diversity Panel wih be used where full genomic structure is available, and ahows screening of the open reading frame of the gene, including sphce junctions. In instances where genomic structure for selected candidate genes may not be available, a cDNA version of the HDP Screening Panel permits screening of the open reading frame of the gene.

This DNA panel is derived from 47 consented individuals from four ethnic groups (Caucasian, African-American, Asian and Hispanic). The panel is sufficiently sized to enable identification of 95% of SNPs with ahele population frequencies >= 5%. Comparable utility of our panel with the NTH Diversity panel was demonstrated by parahel screening of 90 kilobases of coding sequence from each panel.

A cDNA counterpart to our Human Diversity Panel has been generated from lymphoblastoid ceh lines to obviate the need for intron/exon structure in 50% of human genes. In the absence of genomic structure, this methodology wih be employed to screen the entire open reading frame of the gene. Various modifications and variations of the described compositions, methods, and systems of the invention wih be apparent to those skihed in the art without departing from the scope and sphit of the invention. Although the invention has been described in connection with certain embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Nor should the description of such embodiments be considered exhaustive or limit the invention to the precise forms disclosed. Furthermore, elements from one embodiment can be readily recombined with elements from one or more other embodiments. Such combinations can form a number of embodiments within the scope of the invention. It is intended that the scope of the invention be defined by the fohowing claims and their equivalents.

TABLE 1

AACT

Full name : alp a-1-antichymotrypsin

Link : AACT_link_cdna

Subsequence GB:AACT 1 1520 #1

CDS GB:AACT.l 1302 bp #2

ORF 12 1313

Allele GB:AACT 1 36 36 A>G source isSNP SNP00027203 consequence GB:AACT.1 2 Missense 9-9 A>T

Allele GB:AACT 1 269 269 A>G source isSNP SNP00073834 consequence GB:AACT.l 2 Silent 86-! 36 F

Allele GB : ACT 1 830 830 A>G source isSNP SNP00047132 consequence GB:AACT.l 2 Silent 273- -273

Allele GB:AACT 1 836 836 A>G source isSNP SNP00043844 consequence GB:AACT.l 2 Silent 275- -275

Allele GB: ACT 1 837 837 A>G source isSNP SNP00101207 consequence GB:AACT.l 2 Missense 276- -276 F>L

Allele GB :AACT 1 848 848 A>G source isSNP SNP00101208 consequence GB:AACT.1 2 Silent 279- -279

Allele GB : ACT 1 854 854 A>G source isSNP SNP00052361 consequence GB:AACT.1 2 Silent 281- -281

Allele GB.AACT 1 947 947 G>T source isSNP SNP00059862 consequence GB:AACT.1 2 Stop 312- ■312

Allele GB :AACT 1 1227 1227 A>G source isSNP SNP00046872 consequence GB:AACT.l 2 Missense 406- -406 T>A

GIF AACT-cdna-fwd.gif

Link : FL_2114865_link_genomic

Subsequence GB :AL049839_2 1 214520 #3

Subsequence AACT_mrna_build.1 59531 69154 #4

Subsequence AACT_cds.2 59542 67448 #5

CDS AACT_ .eds.2 651 bp 2 exons #5 exon 59542 60184 exon 67441 67448 mRNA AACT_mrna_bι;ιild.1 1523 bp 4 exons #4 exon 59531 60184 exon 64295 64568 exon 67441 67591 exon 68711 69154

Allele GB:AL049839_ 2 3 59566 59566 A>G source isSNP SNP00027203 source wetSNP GB:AL049839_2.v59566.G>A consequence AACT_cds .2 5 Missense 9-9 A>T

Allele GB:AL049839_ 2 3 59799 59799 A>G source isSNP SNP00073834 consequence AACT_cds .2 5 Silent 86-1 36 F

Allele GB:AL049839_ .2 3 59844 59844 A>G TABLE 1 (Cont.) source isSNP SNP00005018 consequence AACT_σds.2 5 Silent 101-101 K

Allele GB:AL049839_2 3 60144 60144 A>G source isSNP SNP00093217 consequence AACT_σds.2 5 Silent 201-201

Allele GB:AL049839_2 3 64470 64470 A>G source isSNP SNP00047132 consequence AACT_cds .2 5 Intron

Allele GB:AL049839_2 3 64476 64476 A>G source isSNP SNP00043844 consequence AACT_cds .2 5 Intron

Allele GB :AL049839_2 3 64477 64477 A>G source isSNP SNP00101207 consequence AACT_cds .2 5 Intron

Allele GB:AL049839_2 3 64488 64488 A>G source isSNP SNP00101208 consequence AACT_cds .2 5 Intron

Allele GB:AL049839_2 3 64494 64494 A>G source isSNP SNP00052361 consequence AACT_cds .2 5 Intron

Allele GB :AL049839_2 3 65434 65434 A>G source isSNP SNP00052361 consequence AACT_cds .2 5 Intron

Allele GB:AL049839_2 3 65440 65440 A>G source isSNP SNP00101208 consequence AACT_cds.2 5 Intron

Allele GB:AL049839_2 3 65451 65451 A>G source isSNP SNP00101207 consequence AACT_cds .2 5 Intron

Allele GB: L049839_2 3 65452 65452 A>G source isSNP SNP00043844 consequence AACT_cds .2 5 Intron

Allele GB :AL049839_2 3 65458 65458 A>G source isSNP SNP00047132 consequence AACT_cds.2 5 Intron

Allele GB:AL049839_2 3 68858 68858 A>G source isSNP SNP00046872 consequence AACT_cds .2 5 3' Allele GB: L049839_2 3 68882 68882 A>G source wetSNP GB:AL049839 2.v68882.A>G consequence AACT_cds .2 5 3 ' GIF AACT-genomic-fwd.gif

ABLl

Full name : v-abl Abelson murine leukemia viral oncogene homolog 1

Link : ABLl_link_cdna

Subsequence GB:NM_005157 5744 #6 CDS GB:NM_005157.1 3393 bp #7

ORF 365 3757 Allele GB:NM_005157 6 1916 1916 OG source isSNP SNP00046020 consequence GB:NM_005157.1 7 Missense 518-518 A>P

Allele GB:NM 005157 6 w 2716 C>G TABLE 1 (Cont.) source isSNP SNP00068702 consequence GB:NM_005157.1 7 Silent 784-784

Allele GB:NM_005157 6 3625 3625 A>G source isSNP SNP00098956 consequence GB:NM_005157.1 7 Silent 1087-1087

Allele GB:NM_005157 6 3688 3688 A>G source isSNP SNP00012765 consequence GB:NM_005157.1 7 Silent 1108-1108

Allele GB:NM_005157 6 3894 3894 C>G source isSNP SNP00046021 consequence GB :NM_005157.1 7 3'

Allele GB:NM_005157 6 4612 4612 A>G source isSNP SNP00051628 consequence GB:NM_005157.1 7 3'

Allele GB:NM_005157 6 5512 5512 A>G source isSNP SNP00012768 consequence GB:NM_005157.1

GIF ABLl-cdna-fwd.gif Link : ABLl_link_genorαic

Subsequence ABLl_cds.l 73887 116507 #8

Subsequence ABLl_cds.2 29132 116507 #9

Subsequenc :e GB:U07561_1 1 35962 #10

Subsequence GB:U07563_1 36063 120601 #11

Subsequenc :e ABLl_mrna_build.1 73506 118495 #12

Subsequence ABLl_mrna_build.2 28792 116507 #13

Subsequenc :e ABLl_mrna_build.3 73724 116507 #14

CDS ABLl_cds.l 3393 bp 11 exons #8 exon 73887 73965 exon 85951 86124 exon 86688 86983 exon 94650 94922 exon 104016 104100 exon 104747 104924 exon 106755 106939 exon 109237 109389 exon 110890 110979 exon 111322 111486 exon 114793 116507

CDS ABLl_cds.2 3450 bp 11 exons #9 exon 29132 29267 exon 85951 86124 exon 86688 86983 exon 94650 94922 exon 104016 104100 exon 104747 104924 exon 106755 106939 exon 109237 109389 exon 110890 110979 exon 111322 111486 exon 114793 116507 mRNA ABLl_mrna_build.l 5762 bp 11 exons #12 exon 73506 73965 exon 85951 86124 exon 86688 86983 exon 94650 94922 TABLE 1 (Cont.) exon 104016 104100 exon 104747 104924 exon 106755 106939 exon 109237 109389 exon 110890 110979 exon 111322 111486 exon 114793 118495 mRNA ABLl_mrna_build.2 3787 bp 11 exons #13 exon 28792 29267 exon 85954 86124 exon 86688 86983 exon 94650 94922 exon 104016 104100 exon 104747 104924 exon 106755 106939 exon 109237 109389 exon 110890 110979 exon 111322 111486 exon 114793 116507 mRNA ABLl_mrna_build.3 3556 bp 11 exons #14 exon 73724 73965 exon 85951 86124 exon 86688 86983 exon 94650 94922 exon 104016 104100 exon 104747 104924 exon 106755 106939 exon 109237 109389 exon 110890 110979 exon 111322 111486 exon 114793 116507

Allele GB:U07561_1 10 29061 29061 A>G source isSNP SNP00120072 consequence ABLl_cds .1 8 5 ' consequence ABLl_cds .2 9 5 '

Allele GB:U07563_1 11 35864 35864 A>G source isSNP SNP00048470 consequence ABLl_cds .1 8 5' consequence ABLl_cds .2 9 9 Intron

Allele GB:U07563_1 11 58876 58876 C>G source wetSNP GB:U07563_l.v58876.OG consequence ABLl_cds .1 8 Intron consequence ABLl_cds .2 9 Intron

Allele GB:U07563_1 11 68640 68640 A>G source wetSNP GB:U07563_1.v68640.T>C consequence ABLl_cds .1 8 ■ Intron consequence ABLl_cds .2 9 Intron

Allele GB:U07563_1 11 74901 74901 A>G source wetSNP GB :U07563 l .v74901 .A>G TABLE 1 (Cont.) consequence ABLl_cds .1 8 Silent 499-499 E consequence ABLl_cds .2 9 Silent 518-518 E

Allele GB:U07563_1 11 75298 75298 C>G source isSNP SNP00046020 consequence ABLl_cds .1 8 Missense 518-518 A>P consequence ABLl_cds .2 9 Missense 537-537 A>P

Allele GB:U07563_1 11 78921 78921 A>G source wetSNP GB:U07563_l.v78921 .G>A consequence ABLl_cds .1 8 Silent 623-623 E consequence ABLl_cds .2 9 Silent 642-642 E

Allele GB:U07563_1 11 79239 79239 A>G source wetSNP GB:U07563_l.v79239 .G>A consequence ABLl_σds .1 8 Silent 729-729 T consequence ABLl_cds .2 9 Silent 748-748 T

Allele GB:U07563_1 11 79404 79404 OG source isSNP SNP00068702 source wetSNP GB:U07563_l.v79404 .OG consequence ABLl_σds .1 8 Silent 784-784 P consequence ABLl_cds .2 9 Silent 803-803 P

Allele GB:U07563_1 11 • 79657 79657 A>G source wetSNP GB:U07563_l.v79657 .OT consequence ABLl_cds .1 8 Missense 869-869 P>S consequence ABLl_cds .2 9 Missense 888-888 P>S

Allele GB:U07563_1 11 79750 79750 A>G source wetSNP GB:U07563_l.v79750 .OT consequence ABLl_cds.1 8 Missense 900-900 P>S consequence ABLl_cds .2 9 Missense 919-919 P>S

Allele GB:U07563_1 11 80313 80313 A>G source isSNP SNP00098956 consequence ABLl_cds .1 8 Silent 1087-1087 I consequence ABLl_cds.2 9 Silent 1106-1106 I

Allele GB:U07563_1 11 80376 80376 A>G source isSNP SNP00012765 source wetSNP GB:U07563_l.v80376 .G>A consequence ABLl_cds .1 8 Silent 1108-1108 P consequence ABLl_cds.2 ^' 9 Silent 1127-1127 P

Allele GB:U07563_1 11 80582 80582 OG source isSNP SNP00046021 consequence ABLl_σds .1 8 3' consequence ABLl_cds .2 9 3'

Allele GB:U07563_1 11 81298 81298 A>G source isSNP SNP00051628 consequence ABLl_σds .1 8 3' consequence ABLl_cds .2 9 3'

Allele GB:U07563_1 11 81806 81806 A>G source isSNP SNP00012766 consequence ABLl_cds .1 8 3' consequence ABLl_cds .2 9 3'

Allele GB:U07563_1 11 82199 82199 A>G source isSNP SNP00012768 consequence ABLl_cds .1 8 3' consequence ABLl_cds .2 9 3'

GIF ABLl-genomic-fwd . gif TABLE 1 (Cont.)

ADAM9

Full name : a disintegrin and metalloproteinase domain 9

Link : ADAM9_link_cdna

Subsequence GB:HSU41766 1 3865 #15

CDS GB-.HSU41766.1 2460 bp #16

ORF 79 2538 Allele GB:HSU41766 15 462 462 G>T source isSNP SNP00060630 consequence GB:HSU41766.1 1 166 M Miisssseennssee 1 12288--112288 I>M

Allele GB:HSU41766 15 1486 1486 AA>>GG source isSNP SNP00122821 consequence GB:HSU41766.1 1 166 M Miisssseennssee 4 47700--447700 G>S

Allele GB:HSU41766 15 1580 1580 G>T source isSNP SNP00060631 consequence GB:HSU41766.1 1 166 M Miisssseennssee 5 50011--550011 N>T

Allele GB:HSU41766 15 2845 2845 A>G source isSNP SNP00024957 consequence GB:HSU41766.1 16 3'

Allele GB:HSU41766 15 3112 3112 A>G source isSNP SNP00122822 consequence GB:HSU41766.1 16 3'

Allele GB:HSU41766 15 3703 3703 A>G source isSNP SNP00024958 consequence GB:HSU41766.1 16

GIF ADAM9 -cdna-fwd.gif

ADAMTSl

Full name : a disintegrin-like and metalloprotease (reprolysin type) with thrombospondin type 1 motif, 1

Link : ADAMTSl_link_cdna

Subsequence GB:AF060152_1 1 3430 #17 CDS GB:AF060152_1.1 2853 bp #18

ORF 238 3090 Allele GB:AF060152_1 17 140 140 OG source isSNP SNP00109009 consequence GB:AF060152_1.1 18 5' Allele GB: F060152_1 17 282 282 G>T source isSNP SNP00071624 consequence GB:AF060152_1.1 18 Silent 15-15- P Allele GB:AF060152_1 17 768 768 G>T source isSNP SNP00069180 consequence GB:AF060152_1.1 1 188 S Siilleenntt 1 17777--117777 V Allele GB :AF060152_1 17 865 865 OG source isSNP SNP00069181 consequence GB:AF060152_1.1 1 188 M Miisssseennssee 2 21100--221100 P>A Allele GB:AF060152_1 17 1686 1686 A>G source isSNP SNP00033973 consequence GB:AF060152_1.1 18 Silent 483-483 Allele GB:AF060152_1 17 2294 2294 A>G source isSNP SNP00109010 consequence GB:AF060152_1.1 1 188 M Miisssseennssee 6 68866--668866 R>H Allele GB :AF060152_1 17 2370 2370 A>G source isSNP SNP00033<174 TABLE 1 (Cont.) consequence GB:AF060152_1.1 18 Silent 711-711 Allele GB:AF060152_1 17 2958 2958 A>G source isSNP SNP00033975 consequence GB:AF060152_1.1 18 Silent 907-907 GIF ADAMTSl-cdna-fwd.gif

ADAMTS4

Full name : a disintegrin-like and metalloprotease (reprolysin type) with thrombospondin type 1 motif, 4

Link : ADAMTS4_link_cdna

Subsequence GB:NM_005099_1 1 4301 #19 CDS GB:NM_005099_1.1 2514 bp #20

ORF 401 2914 Allele GB:NM_005099_1 19 2970 2970 A>G source isSNP SNP00022951 consequence GB:NM_005099_1.1 20 3' Allele GB:NM_005099_1 19 3529 3529 A>G source dbSNP gnl | dbSNP| ss610462_allele consequence GB:NM_005099_1.1 20 3' Allele GB:NM_005099_1 19 3533 3533 A>G source dbSNP gnl | dbSNP | ss722414_allele source dbSNP gnl j dbSNP j ss999631_allele consequence GB:NM__005099_1.1 20 3' Allele GB:NM_005099_1 19 3855 3855 A>G source dbSNP gnl | dbSNP| ssl298908_allele consequence GB:NM_005099_1.1 20 3' GIF ADAMTS4-cdna-fwd.gif

AGCl

Full name : aggrecan 1

Link : AGCl__link_cdna

Subsequence GB:HUMAGPRO 1 7137 #21 CDS GB:HUMAGPR0.1 6951 bp #22

ORF 61 7011

Allele GB:HUMAGPRO 21 6495 6495 G>T source isSNP SNP00010327 consequence GB :HUMAGPRO.1 22 Silent 2145-2145 A

GIF AGC1- -cdna-fwd.gif

ANK

Full name : human homolog of mouse ank gene

Link : ANK_fl_link_cdna

Subsequence FN: 3255641CB1 1 1481 #23 CDS FN:3255641CB1.1 1338 bp #24

ORF 106 1443

Allele FN:3255641CB1 23 258 258 A>G source disSNP SNP00073561 consequence FN:3255641CB1.1 24 Silent 51-51 A

Allele FN: 3255641CB1 23 1048 1048 OG TABLE 1 (Cont.) source isSNP SNP00036339 consequence FN: 3255641CB1.1 24 Missense 315-315 A>P

Allele FN: 3255641CB1 23 1106 1106 A>G source isSNP SNP00075037 consequence FN: 3255641CB1.1 24 Missense 334-334 V>A

Allele FN:3255641CBl 23 1373 1373 A>G source isSNP SNP00045819 consequence FN: 3255641CB1.1 24 Missense 423-423 S>F

GIF ANK-cdna-fwd.gif Link : ANK_link_cdna

Subsequence GB:AF274753_1 1 1568 #25

CDS GB:AF274753_1.1 1479 bp #26 ORF 69 1547

Allele GB:AF274753_1 25 362 362 A>G source isSNP SNP00073561 consequence GB : :AF274753_1.1 26 Silent 98-98 A

Allele GB:AF274753_1 25 1152 1152 OG source isSNP SNP00036339 consequence GB: :AF274753_1.1 26 Missense 362-362 A>P

Allele GB:AF274753_1 25 1210 1210 A>G source isSNP SNP00075037 consequence GB : :AF274753_1.1 26 Missense 381-381 V>A

Allele GB:AF274753_1 25 1477 1477 A>G source isSNP SNP00045819 consequence GB : :AF274753_1.1 26 Missense 470-470 S>F

GIF ANK-cdna-fwdl.gif

.nk : ANK_ _.1ink_genomic

Subsequence ANK_cds .1 26332 84281 #27

Subsequence GBI:AC016575 >_6_000010 1 605 #28

Subsequence GB:AC026437_ .2 706 92528 #29

Subsequence ANK_mrna_build.1 308 85658 #30

Subsequence ANK_σds .2 272 84281 #31

CDS ANK_cds.l 1338 bp 11 exons #27 exon 26332 26503 exon 36882 37000 exon 39535 39618 exon 44240 44410 exon 46173 46307 exon 49517 49609 exon 53557 53652 exon 78643 78772 exon 81811 81934 exon 82505 82604 exon 84168 84281

CDS ANK_c :ds.2 1479 bp 12 exons #31 exon 272 367 exon 26287 26503 exon 36882 37000 exon 39535 39618 exon 44240 44410 exon 46173 46307 exon 49517 49609 exon 53557 53652 exon 78643 78772 exon 81811 81934 TABLE 1 (Cont.) exon 82505 82604 exon 84168 84281 mRNA AN_mrna_buiId.1 2820 bp 12 exons #30 exon 308 367 exon 26287 26503 exon 36882 37000 exon 39535 39618 exon 44240 44410 exon 46173 46307 exon 49517 49609 exon 53557 53652 exon 78643 78772 exon 81811 81934 exon 82505 82604 exon 84168 85658

Allele GB:AC026437_2 29 8413 8413 OG source dbSNP gnl | dbSNP| ss95678_allele consequence ANK_cds .1 27 5' consequence ANK_cds .2 31 Intron

Allele GB:AC026437_2 29 14825 14825 A>G source dbSNP gnl|dbSNP|ss619053_allele source dbSNP gnljdbSNP|ssl002004_allele source dbSNP gnljdbSNP|ss227983_allele source dbSNP gnljdbSNP|ss324626_allele consequence ANK_cds .1 27 5' consequence ANK_cds .2 31 Intron

Allele GB:AC026437.2 29 25779 25779 A>G source wetSNP GB:AC026437_2.v25779.OT consequence ANK_cds .1 27 Silent 51-51 A consequence AN_cds .2 31 Silent 98-98 A

Allele GB:AC026437_2 29 25807 25807 A>G source isSNP SNP00104502 source wetSNP GB:AC026437_2.v25807.G>A consequence ANK_cds .1 27 Intron consequence ANK_cds.2 31 Intron

Allele GB:AC026437_2 29 26433 26433 A>G source isSNP SNP00018441 consequence ANK_cds .1 27 Intron consequence ANK_cds.2 31 Intron

Allele GB:AC026437_2 29 30696 30696 A>T source dbSNP gnl | dbSNP | ssl016631_allele source dbSNP gnl j bSNP| ss389763_allele consequence ANK_cds .1 27 Intron consequence ANK_cds.2 31 Intron

Allele GB:AC026437_2 29 34277 34277 A>G source isSNP SNP00101566 consequence ANK_cds .1 27 Intron consequence ANK_cds .2 31 Intron

Allele GB:AC026437_2 29 36172 36172 A>G source wetSNP GB:AC026437_2.v36172.T>C consequence ANK_cds .1 27 Intron consequence ANK_cds.2 31 Intron

Allele GB:AC026437_2 29 37028 37028 G>T source isSNP SNP00056800 consequence ANK eds .1 Intron

²1⁷50 TABLE 1 (Cont.) consequence ANK_cds.2 31 Intron

Allele GB :AC026437..2 29 37186 37186 G>T source isSNP SNP00022144 consequence ANK_cds.l 27 Intron consequence ANK_cds.2 31 Intron

Allele GB:AC026437._2 29 37205 37205 A>G source isSNP SNP00022143 consequence ANK_cds.l 27 Intron consequence ANK_cds.2 31 Intron

Allele GB:AC026437.2 29 37340 37340 A>T source dbSNP gnl|dbSNP|ss469809_allele consequence AN_cds .1 27 Intron consequence ANK_cds .2 31 Intron

Allele GB:AC026437.2 29 52817 52817 G>T source wetSNP GB:AC026437_2.v52817. C>A consequence ANK_cds .1 27 Intron consequence ANK_cds .2 31 Intron

Allele GB:AC026437._.2 29 52899 52899 A>G source wetSNP GB:AC026437_2.v52899.A>G consequence ANK_cds .1 27 Silent 274-274 A consequence ANK_cds .2 31 Silent 321-321 A

Allele GB:AC026437.2 29 52962 52962 G>T source wetSNP GB:AC026437_2.v52962.T>G consequence ANK_cds .1 27 Intron consequence ANK_cds .2 31 Intron

Allele GB:AC026437.2 29 63950 63950 A>G source isSNP SNP00093702 consequence AN _cds.l . ..27 Intron consequence ANK_cds.2... 31. Intron

Allele GB:AC026437.2 29 78010 78010 OG source isSNP SNP00036339 consequence ANK_cds.l 27 Missense 315-315 A>P consequence AN_cds.2 31 Missense 362-362 A>P

Allele GB:AC026437.2 29 78875 78875 A>G source isSNP SNP00095793 consequence ANK_cds .1 27 Intron consequence ANK_cds .2 31 Intron

Allele GB:AC026437.2 29 81235 81235 A>G source wetSNP GB:AC026437_2.v81235.T>C consequence AN_cds .1 27 Intron consequence ANK_cds .2 31 Intron

Allele GB:AC026437.2 29 82852 82852 A>G source isSNP SNP00120424 consequence ANK_cds.l 27 Intron consequence ANK_cds.2 31 Intron

Allele GB:AC026437..2 29 83057 83057 A>G source isSNP SNP00120425 consequence ANK_cds.l 27 Intron consequence ANK_cds.2 31 Intron

Allele GB:AC026437.2 29 83506 83506 A>G source isSNP SNP00045819 consequence ANK_cds.l 27 Missense 423-423 S>F consequence ANK_cds.2 31 Missense 470-470 S>F

Allele GB:AC026437.2 29 83587 83587 A>G source wetSNP >ι AC026437 2.v83587.G>A TABLE 1 (Cont.) consequence ANK_cds.l 27 3' consequence ANK_cds.2 31 3'

Allele GB:AC026437.2 29 83607 83607 A>G source isSNP SNP00008779 source wetSNP GB:AC026437_2.v83607.A>G consequence ANK_σds.l 27 3' consequence ANK_cds.2 31 3'

Allele GB:AC026437. .2 29 84086 84086 A>G source isSNP SNP00012596 consequence ANK_cds.l 27 3' consequence ANK_cds.2 31 3'

Allele GB:AC026437. .2 29 84156 84156 A>G source isSNP SNP00045820 consequence ANK_cds.l 27 3' consequence ANK_cds.2 31 3'

Allele GB:AC026437. .2 29 84896 84896 G>T source isSNP SNP00045822 consequence AN_cds.l 27 consequence ANK_cds.2 31

GIF ANK-genomic-fwd.gif

BGLAP

Full name : Bone Gla Protein

Link : FL_104137_link_genomic

Subsequence GB:AC007227_2_104137CD1 35521 34594 #32 Subsequence GB :AC007227_2 1 167932 #33 Subsequence BGLAP_mrna_build.l 35539 34461 #34 mRNA BGLAP_mrna_build.l 451 bp 44 eexxoonnss #34 exon 35539 35458 exon 35200 35162 exon 34991 34922 exon 34720 34461

CDS GB:AC007227_2_104137CD1 300 bp 4 exons #32 exon 35521 35458 exon 35200 35162 exon 34991 34922 exon 34720 34594 Allele GB:AC007227_2 33 34618 34618 OG source wetSNP GB:AC007227_2. 34618.G>C consequence GB:AC007227_2_104137CD1 32 Silent 92-92 A

Allele GB:AC007227_2 33 34977 34977 G>T source wetSNP GB:AC007227_2.v34977.G>T consequence GB:AC007227_2_104137CD1 32 Missense 40-40 Q>

Allele GB:AC007227_2 33 35228 35228 OG source isSNP SNP00038471 consequence GB:AC007227_2_104137CD1 32 Intron

GIF BGLAP-genomic-rev.gif

BGN

Full name : BGN

Link : BGN_link_cdna TABLE 1 (Cont.)

Subsequence GB :HUMHPGI 1685 #35 CDS GB:HUMHPGI. 1107 bp #36

ORF 121 1227 Allele GB:HUMHPGI 35 70 70 G>T source isSNP SNP00011488 consequence GB:HUMHPGI.1 36 5'

Allele GB:HUMHPGI 35 261 261 A>G source isSNP SNP00011489 consequence GB:HUMHPGI.1 36 Silent 47-47 S

Allele GB:HUMHPGI 35 660 660 A>G source isSNP SNP00011490 consequence GB:HUMHPGI.1 36 Silent 180-180

Allele GB:HUMHPGI 35 1355 1355 A>G source isSNP SNP00092805 consequence GB:HUMHPGI.1 36 GIF BGN-cdna-fwd.gif Link : BGN_link_genomic

Subsequence GB:U82695 1 76146 #37 Subsequence GB:U82695_2540367CD1 18042 21854 #38 Subsequence BGN_mrna_build.1 8415 22311 #39 CDS GB:U82695_2540367CD1 1107 bp 7 exons #38 exon 18042 18279 exon 18648 18760 exon 19272 19485 exon 19938 20048 exon 20239 20332 exon 20456 20594 exon 21657 21854 mRNA BGN_mrna_build.l 1684 bp 8 exons #39 exon 8415 8523 exon 18031 18279 exon 18648 18760 exon 19272 19485 exon 19938 20048 exon 20239 20332 exon 20456 20594 exon 21657 22311

Allele GB:U82695 37 8484 8484 G>T source isSNP SNP00011488 consequence GB:U82695_2540367CD1 38 5'

Allele GB:U82695 37 18161 18161 A>G source wetSNP GB:U82695.vl8161.A>G consequence GB:U82695_2540367CD1 38 Silent 40-40 E

Allele GB:U82695 37 18182 18182 A>G source isSNP SNP00011489 source wetSNP GB:U82695.vl8182.G>A consequence GB:U82695_2540367CD1 38 Silent 47-47 S

Allele GB:U82695 37 18330 18330 A>G source wetSNP GB:U82695.vl8330.G>A consequence GB:U82695_2540367CD1 38 Intron

Allele GB:U82695 37 18354 18354 A>G source wetSNP GB:U82695.vl8354 -G>A consequence GB:U82695_2540367CD1 38 Intron

Allele GB:U82695 37 19460 19460 A>G source isSNP ΞNP00011490 TABLE 1 (Cont.) source wetSNP GB:U82695.vl9460.T>C consequence GB:U82695_2540367CD1 38 Silent 180-180 s

Allele GB:U82695 37 21566 21566 G>T source wetSNP GB:U82695.v21566.G>T consequence GB:U82695_2540367CD1 38 Intron

Allele GB:U82695 37 21639 21639 A>G source wetSNP GB:U82695.v21639.C>T consequence GB:U82695_2540367CD1 38 Intron

Allele GB:U82695 37 21982 21982 A>G source isSNP SNP00092805 consequence GB:U82695_2540367CD1 38

Allele GB:U82695 37 22172 22172 OT source isSNP SNP00011491 consequence GB:U82695 2540367CD1 38

GIF BGN- -genomic-fwd. gi

BHLHB2

Full name : basic helix-loop-helix domain containing, class B,

Link : BHLHB2_link_cdna

Subsequence GB:AB004066_1 1 2922 #40 CDS GB:AB004066_1.1 1239 bp #41

ORF 197 1435 Allele GB:AB004066_ 40 196 196 A>G source isSNP SNP00062724 consequence GB:AB004066_1.1 41 5'

Allele GB:AB004066_ 1 40 829 829 A>G source isSNP SNP00046376 consequence GB:AB004066_1.1 41 Silent 211-211

Allele GB:AB004066_.1 40 2070 2070 A>G source isSNP SNP00013041 consequence GB:AB004066_1.1 41 3'

Allele GB:AB004066..1 40 2323 2323 A>G source isSNP SNP00013042 consequence GB:AB004066_1.1 41

GIF BHLHB2-cdna-fwd.gif

BMP2

Full name : BMP2

Link : BMP2_link_cdna

Subsequence GB :HUMBMP2A 1547 #42

CDS GB:HUMBMP2A.l 1191 bp #43

ORF 324 1514

Allele GB :HUMBMP2A 42 584 584 A>G source isSNP SNP00015730 consequence GB:HUMBMP2A.l 43 Silent 87-87 S

Allele GB:HUMBMP2A 42 760 760 A>G source isSNP SNP00015731 consequence GB:HUMBMP2A.l 43 Missense 146-146 T>I

Allele GB:HUMBMP2A 42 984 984 G>T source isSNP SNP0001JL32 TABLE 1 (Cont.) consequence GB:HUMBMP2A.l 43 Missense 221-221 H>N Allele GB:HUMBMP2 42 1484 1484 A>G source isSNP SNP00015733 consequence GB:HUMBMP2A.l 43 , Silent 387-387 D GIF BMP2-cdna-fwd.gif Link : FL_3220019_link_genomic

Subsequence GB:HS859D4 1 178870 #44 Subsequence GB:HS859D4_3220019CD1 176685 167723 #45

Subsequence BMP2 mrna build.1 178252 167687 #46 mRNA BMP2_mrna_b ild.l 1547 bp 3 exons #46 exon 178252 177937 exon 176692 176340 exon 168564 167687 CDS GB:HS859D4_3220019CD1 1188 bp 2 exons #45 exon 176685 176340 exon 168564 167723 Allele GB:HS859D4 44 167750 167750 A>G source isSNP SNP00015733 consequence GB:HS859D4_3220019CD1 45 Silent 387-387

D

Allele GB:HS859D4 44 168250 168250 G>T source isSNP SNP00015732 consequence GB:HS859D4 3220019CD1 45 Missense 221-221

H>N Allele GB:HS859D4 44 168341 168341 A>T source wetSNP GB:HS859D4.vl68341.T>A consequence GB:HS859D4 3220019CD1 45 Missense 190-190

R>S Allele GB:HS859D4 44 168474 168474 A>G source isSNP SNP00015731 consequence GB:HS859D4_3220019CD1 45 Missense 146-146

T>I Allele GB:HS859D4 44 176425 176425 A>G source isSNP SNP00015730 source wetSNP GB:HS859D4.vl76425.T>C consequence GB:HS859D4 3220019CD1 45 Silent 87-87 S

GIF BMP2-genomic-rev.gif

BMP4

Full name : BMP4

Link : BMP4_link_cdna

Subsequence GB:HUMBMP2B 1751 #47

CDS GB :HUMBMP2B .1 1227 bp #48

ORF 395 1621 Allele GB:HUMBMP2B 47 308 308 A>G source isSNP SNP00074676 consequence GB:HUMBMP2B.l 48

Allele GB:HUMBMP2B 47 849 849 A>G source isSNP SNP00000573 consequence GB:HUMBMP2B.l 48 Missense 152-152 V>A

GIF BMP4-cdna-fwd.gif Link : BMP4_link_genomic

Subsequence GB:HSU43842 33 #49 tø TABLE 1 (Cont.)

Subsequence GB:HSU43842_1613615CD1 7798 9984 #50 Subsequence BMP4 mrna build.1 3207 10117 #51 mRNA BMP4_mrna_build.1 1751 bp 4 exons #51 exon 3207 3468 exon 6620 6744 exon 7791 8167 exon 9131 10117

CDS GB:HSU43842_1613615CD1 1224 bp 2 exons #50 exon 7798 8167 exon 9131 9984 Allele GB:HSU43842 49 6665 6665 A>G source isSNP SNP00074676 consequence GB:HSU43842_1613615CD1 50 5'

Allele GB:HSU43842 49 7752 7752 A>G source isSNP SNP00117542 consequence GB:HSU43842_1613615CD1 50 5'

Allele GB:HSU43842 49 9215 9215 A>G source isSNP SNP00000573 source wetSNP GB:HSU43842.v9215.C>T consequence GB:HSU43842_1613615CD1 50 Missense 152-152 A>V

GIF BMP4-genomic-fwd.gif

BMP6

Full name : BMP6

Link : BMP6_link_cdna

Subsequence GB :HUMTGFBC 2923 #52

CDS GB:HUMTGFBC.1 1542 bp #53

ORF 160 1701

Allele GB:HUMTGFBC 52 1263 1263 OG source isSNP SNP00069306 consequence GB:HUMTGFBC.1 53 Silent 368-368 V

Allele GB:HUMTGFBC 52 2280 2280 G>T source isSNP SNP00021640 consequence GB:HUMTGFBC.1 53

Allele GB:HUMTGFBC 52 2436 2436 A>G source isSNP SNP00003240 consequence GB:HUMTGFBC.1 53

Allele GB :HUMTGFBC 52 2574 2574 A>G source isSNP SNP00021639 consequence GB :HUMTGFBC.1 53

GIF BMP6- -cdna-fwd.gif

CAPN4

Full name : calpain, small polypeptide

Link : FL_508926_link_genomic

Subsequence GB:CH19F24590 1 41369 #54 Subsequence GB:CH19F24590_3639962CD1 31006 39830 #55 Subsequence FL_3639962_mrna_build.1 30073 40241 #56 Subsequence CAPN4_cds .1 31006 39833 #57 mRNA FL_3639962_mrna_b ild.l 1309 bp 11 exons #56 TABLE 1 (Cont.) exon 30073 30151 exon 30991 31214 exon 32294 32327 exon 32646 32735 exon 32903 32960 exon 33058 33122 exon 35800 35868 exon 35970 36048 exon 36190 36306 exon 39572 39630 exon 39807 40241

CDS CAPN4. _cds .1 717 bp 9 exons #57 exon 31006 31214 exon 32294 32327 exon 32903 32960 exon 33058 33122 exon 35800 35868 exon 35970 36048 exon 36190 36306 exon 39572 39630 exon 39807 39833

CDS GB:CH19F24590_3639962CD1 8 804 bp 10 exons #55 exon 31006 31214 exon 32294 32327 exon 32646 32735 exon 32903 32960 exon 33058 33122 exon 35800 35868 exon 35970 36048 exon 36190 36306 exon 39572 39630 exon 39807 39830

GIF CAPN4- -genomic-fwd.gif

CBFAl

Full name : CBFAl

Link : CBFAl_link_cdna

Subsequence GB : HUMCBFA 1 1411 #58

CDS GB : HUMCBFA . 2 1323 bp #59

ORF 1 1323

Allele GB : HUMCBFA 58 260 260 A>G source isSNP SNP00063798 consequence GB :HUMCBF .2 59 Missense 87-87 G>E GIF CBFAl-cdna-fwd . gif Link : CBFAl_link_genomic

Subsequence GB:HSCBFA1S1 1 93 #60 Subsequence GB-.HSCBFA1S2 194 669 #61 Subsequence GB:HSCBFA1S3 770 1034 #62 Subsequence GB:HSCBFA1S4 1135 1381 #63 Subsequence GB:HSCBFA1S5 1482 1759 #64 Subsequence GB:HSCBFA1S6 1860 2081 #65 Subsequence GB:HSCBFA1S7 2182 2301 #66 Subsequence GB:HSCBFA1S8 2402 3033 #67 TABLE 1 (Cont.)

Subsequence CBFAl_cds.l 28 2948 #68 CDS CBFAl_cds. 1566 bp 8 exons #68 exon 28 85 exon 261 625 exon 821 977 exon 1198 1302 exon 1533 1706 exon 1881 2042 exon 2201 2266 exon 2470 2948 Allele GB:HSCBFA1S3 62 177 177 A>G source wetSNP GB :HSCBFA1S3.vl77. OT consequence CBFAl_cds .1 68 Silent 183-183 N

Allele GB:HSCBFA1S8 67 490 490 A>G source wetSNP GB :HSCBFA1S8.v490. OT consequence CBFAl_cds .1 68 Silent 503-503 GIF CBFAl-genomic-fwd.gif

CD36

Full name : CD36 Glycoprotein

Link : CD36 link cdna

Subsequence EM:HSCD3621 1 2216 #69

Allele EM:HSCD3621 69 123 123 OT source isSNP SNP00011023

Allele EM:HSCD3621 69 196 196 A>G source isSNP SNP00096573

Allele EM:HSCD3621 69 230 230 OG source isSNP SNP00110263

Allele EM:HSCD3621 69 827 827 A>G source isSNP SNP00115780

Allele EM:HSCD3621 69 1332 1332 A>G source isSNP SNP00096574

Link : CD36_link_genomic

Subsequence CD36_link_cds.l 2094 6548 #70

Subsequence EM:HSCD36G1 101 236 #71

Subsequence EM:HSCD36A 338 2898 #72

Subsequence EM:HSCD36G4 3000 3220 #73

Subsequence EM:HSCD36G5 3322 3529 #74

Subsequence EM:HSCD36AA 3631 3999 #75

Subsequence EM:HSCD36G7 4101 4252 #76

Subsequence EM:HSCD36G8 4354 4460 #77

Subsequence EM:HSCD36G9 4562 4691 #78

Subsequence EM:HSCD36G10 4793 5042 #79

Subsequence EM:B74110 5144 5803 #80

Subsequence EM:HSCD36G12 5905 6038 #81

Subsequence EM:HSCD36G13 6140 6252 #82

Subsequence EM:HSCD36G14 6354 6847 #83

Subsequence EM:HSCD36G15 6949 7632 #84

Subsequence CD36_mrna_build.1 136 7602 #85 mRNA CD36. _mrna_build.1 2217 bp 16 exons #85 exon 136 206 exon 1446 1539 exon 2005 2213 TABLE 1 (Cont.) exon 3030 3190 exon 3352 3499 exon 3719 3898 exon 4131 4222 exon 4384 4430 exon 4592 4661 exon 4824 5011 exon 5265 5383 exon 5935 6008 exon 6168 6222 exon 6384 6548 exon 6979 7071 exon 7152 7602

CDS CD36_link_cds.l 1419 bp 12 exons #70 exon 2094 2213 exon 3030 3190 exon 3352 3499 exon 3719 3898 exon 4131 4222 exon 4384 4430 exon 4592 4661 exon 4824 5011 exon 5265 5383 exon 5935 6008 exon 6168 6222 exon 6384 6548

Allele EM:HSCD36A 72 1160 1160 G>T source isSNP SNP00011023 consequence CD36_link_cds.l 70 5'

Allele EM:HSCD36A 72 1698 1698 A>G source isSNP SNP00096573 consequence CD36_link_cds.l 70 5'

Allele EM:HSCD36A 72 1732 1732 OG source isSNP SNP00110263 consequence CD36_link_σds.l 70 5'

Allele EM:HSCD36G4 73 102 102 OG source wetSNP EM:HSCD36G4.vl02.G>C consequence CD36_link_cds.l 70 Missense 64-64 Q Q>H

Allele EM:HSCD36AA 75 232 232 A>G source isSNP SNP00115780 consequence CD36_link_cds .1 70 Silent 191-191

Allele EM:HSCD36G10 79 92 92 A>G source wetSNP EM:HSCD36G1Cl.v92.T>C consequence CD36_link_cds.l 70 Silent 293-293

Allele EM:B74110 80 193 193 A>G source isSNP SNP00096574 consequence CD36_link_cds .1 70 Silent 360-360

Allele EM:HSCD36G14 83 198 203 AAGTAT>AT source wetSNP EM-.HSCD36G14 = •vl98.AAGTAT>AT consequence CD36_link_cds.l 70 3'

Allele EM:HSCD36G14 83 421 421 A>G source isSNP SNP00041723 consequence CD36_link_cds.l 70 3'

GIF CD36-genomic-fwd. gif TABLE 1 (Cont.)

CD68

Full name : CD68 antigen

Link : FL_3777141_link_cdna

Subsequence FN:3777141CB1 1558 #86 CDS FN:3777141CB1.1 1065 bp #87

ORF 75 1139 Allele FN:3777141CB1 86 834 834 G>T source isSNP SNP00006442 consequence FN: 3777141CB1.1 87 Missense 254-254 Q>

Allele FN:3777141CB1 86 1394 1394 G>T source dbSNP gnl|dbSNP|ss450666_allele consequence FN: 3777141CB1.1 87 3' Allele FN:3777141CB1 86 1475 1475 G>T source isSNP SNP00108664 consequence FN: 3777141CB1.1 87 GIF CD68-cdna~fwd.gif Link : FL_1803929_link_genomic

Subsequence GB:AC007421_12 1 95240 #88 Subsequence GB:AC007421_12_3777141CDl 92493 90660 #89 Subsequence FL_3777141_mrna_build.1 92567 90242 #90 mRNA FL_3777141_mrna_build.1 1557 bp 6 exons #90 exon 92567 92445 exon 92361 91844 exon 91705 91586 exon 91460 91388 exon 91275 91105 exon 90793 90242

CDS GB:AC007421_ 12_3777141CD1 1065 bp 6 exons #89 exon 92493 92445 exon 92361 91844 exon 91705 91586 exon 91460 91388 exon 91275 91105 exon 90793 90660

Allele GB:AC007421_12 88 90404 90404 G>T source dbSNP gnl|dbSNP|ss450666_allele consequence GB:AC007421_12_3777141CD1 89 3'

Allele GB:AC007421..12 88 90707 90707 A>G source wetSNP GB:AC007421_12.v90707. T consequence GB:AC007421 12_3777141CDl 89 Missense

340-340 A>T Allele GB:AC007421 12 88 91388 91388 G>T source etSNP GB:AC007421_12.v91388.G>T consequence GB:AC007421_12_3777141CD1 89 Missense

254-254 Q> Allele GB:AC007421_12 88 92357 92357 A>G source wetSNP GB:AC007421_12. 92357.OT consequence GB:AC007421_12_3777141CD1 89 Silent 18-18 Q

GIF CD68-genomic-rev.gif

CDOl TABLE 1 (Cont.)

Full name : cysteine dioxygenase type I

Link : CDOl_link_cdna

Subsequence GB:HHSCYSDIO 1 1556 #91 CDS GB:HHSCYSDI0.1 603 bp #92

ORF 255 857 Allele GB:HHSCYSDIO 91 100 100 A>G source isSNP SNP00009024 consequence GB:HHSCYSDIO.1 92 5'

Allele GB:HHSCYSDIO 91 737 737 A>G source isSNP SNP00048574 consequence GB:HHSCYSDIO.1 92 Silent 161-161

Allele GB:HHSCYSDIO 91 784 784 A>G source isSNP SNP00036859 consequence GB :HHSCYSDIO.1 92 Missense 177-177 V>A

Allele GB:HHSCYSDIO 91 1082 1082 A>G source isSNP SNP00107326 consequence GB :HHSCYSDIO.1 92 3'

Allele GB:HHSCYSDIO 91 1525 1525 A>G source isSNP SNP00036860 consequence GB:HHSCYSDIO.1 92 GIF CDOl-cdna-fwd.gif Link : CD01_link_genomic

Subsequence CD01_cds .1 1653 4275 #93

Subsequence GB:D85778_1 1 2601 #94

Subsequence GB:D85779_1 2702 2938 #95

Subsequence GB:D85780_1 3039 3525 #96

Subsequence GB:D85781_1 3626 4090 #97

Subsequence GB:D85782_1 4191 4921 #98

Subsequence CDOl_mrna_buιild.1 1402 4921 #99 mRNA CDOl_mrna_build.1 1500 bp 5 exons #99 exon 1402 1822 exon 2789 2866 exon 3178 3332 exon 3777 3946 exon 4246 4921

CDS CDOl_c :ds.l 603 bp 5 exons #93 exon 1653 1822 exon 2789 2866 exon 3178 3332 exon 3777 3946 exon 4246 4275

Allele GB:D85778_1 94 1498 1498 A>G source isSNP SNP00009024 consequence CD01_cds.l 93 5'

Allele GB:D85781_1 97 278 278 A>G source isSNP SNP00036859 consequence CD01_cds.l 93 Missense 177-177 V>A

Allele GB:D85782_1 98 310 310 A>G source isSNP SNP00107326 consequence CD01_cds.l 93 3'

GIF CD01-genomic-fwd.gif

CGI-52 TABLE 1 (Cont.)

Link : CGI-52_link_cdna

Subsequence GB :AF151810 1 1414 #100

CDS GB:AF151810.1 1080 bp #101

ORF 277 1356 Allele GB:AF151810 100 1335 1335 A>G source isSNP SNP00054191 consequence GB:AF151810.1 101 Silent 353-353 GIF CGI-52-cdna-fwd.gif Link : CGI-52_link_genomic

Subsequence GB:AC023176_7 1 193672 #102 Subsequence CGI-52_mrna_build.1 131456 93050 #103 mRNA CGI-52 mrna build.1 1420 bp exons #103 exon 131456 131084 exon 119505 119186 exon 97592 97445 exon 96844 96741 exon 96095 95978 exon 93964 93912 exon 93353 93050

Allele GB:AC023176_7 102 93129 93129 A>G source isSNP SNP00054191 Allele GB:AC023176_7 102 93416 93416 A>G source i'sSNP SNP00057212 Allele GB:AC023176_7 102 131305 131305 OG source isSNP SNP00069496 GIF CGI-52-genomic-rev.gif

CHI3L1

Full name : chitinase 3-like 1

Link : CHI3Ll_link_cdna

Subsequence GB:NM_001276_1 1925 #104 CDS GB:NM_001276_1.1 1152 bp #105

ORF 127 1278 Allele GB:NM_001276_1 104 559 559 A>G source isSNP SNP00008252 consequence GB:NM_001276_1.1 105 Missense 145-145 R>G

Allele GB:NM_001276_1 104 590 590 A>G source isSNP SNP00071935 consequence GB:NM_001276_1.1 105 Missense 155-155 K>R

Allele GB:NM_001276_1 104 646 646 G>T source isSNP SNP00022932 consequence GB:NM_001276_1.1 105 Missense 174-174 L>I

Allele GB:NM_001276_1 104 1300 1300 A>G source isSNP SNP00052666 consequence GB:NM__001276_1.1 105 3'

Allele GB:NM_001276_1 104 1342 1342 A>G source isSNP SNP00072805 consequence GB:NM_001276_1.1 105 3'

Allele GB:NM_001276_1 104 1739 1739 A>G source isSNP SNP00076686 consequence GB:NM_001276_1.1 105

GIF CHI3Ll-cdna-fwd.gif

Link : CHI3Ll_link_genomic TABLE 1 (Cont.)

Subsequenc :e CHI3L1 _cds .1 1295 7276 #106

Subsequenc :e CHI3L1. _cds . 1295 7433 #107

Subsequence CHI3L1 _cds .3 1295 7276 #108

Subsequence CHI3L1 _cds .4 1295 2802 #109

Subsequence GB:Y08374_1 1 1635 #110

Subsequence GB:Y08375_1 1736 3186 #111

Subsequenc :e GB:Y08376_1 3287 4116 #112

Subsequence GB:Y08377_1 4217 5035 #113

Subsequence GB:Y08378_1 5136 7923 #114

Subsequence CHI3L1. _mrna_build. ,1 1169 7923 #115

Subsequenc :e CHI3L1. _mrna_bui1d. ,2 1169 7604 #116 mRNA CHI3Ll_mrna_build .2 1355 bp 11 exons #116 exon 1169 1319 exon 1572 1601 exon 2036 2237 exon 2789 2845 exon 3606 3756 exon 4517 4638 exon 5436 5559 exon 6069 6251 exon 6844 6960 exon 7296 7456 exon 7548 7604

CDS CHI3Ll_cds. 1 1152 bp 10 exons #106 exon 1295 1319 exon 1572 1601 exon 2036 2237 exon 2789 2845 exon 3606 3756 exon 4517 4638 exon 5436 5559 exon 6069 6251 exon 6844 6960 exon 7136 7276

CDS CHI3Ll_cds. 2 1149 bp 10 exons #107 exon 1295 1319 exon 1572 1601 exon 2036 2237 exon 2789 2845 exon 3606 3756 exon 4517 4638 exon 5436 5559 exon 6069 6251 exon 6844 6960 exon 7296 7433

CDS CHI3Ll_cds. 3 969 bp 9 exons #108 exon 1295 1319 exon 1572 1601 exon 2036 2237 exon 2789 2845 exon 3606 3756 exon 4517 4638 exon 5436 5559 exon 6844 6960 exon 7136 7276 TABLE 1 (Cont.) mRNA CHI3Ll_mrna_build.1 1925 ]op 10 exons #115 exon 1169 1319 exon 1572 1601 exon 2036 2237 exon 2789 2845 exon 3606 3756 exon 4517 4638 exon 5436 5559 exon 6069 6251 exon 6844 6960 exon 7136 7923

CDS CHI3Ll_cds.4 69 bp 3 exons #109 exon 1295 1319 exon 1572 1601 exon 2789 2802

Allele GB:Y08376_1 112 311 311 G>T source isSNP SNP00071934 consequence CHI3Ll_cds.l 106 Intron consequence CHI3Ll_cds.2 107 Intron consequence CHI3Ll_cds.3 108 Intron consequence CHI3Ll_cds.4 109 3'

Allele GB:Y08376_1 112 438 438 A>G source isSNP SNP00008252 consequence CHI3Ll_cds.l 106 Missense 145- -145 R>G consequence CHI3Ll_cds.2 107 Missense 145- -145 R>G consequence CHI3Ll_cds.3 108 Missense 145- -145 R>G consequence CHI3Ll_cds.4 109 3'

Allele GB:Y08377_1 113 355 355 G>T source isSNP ΞNP00022932 consequence CHI3Ll_cds.l 106 Missense 174- -174 L>I consequence CHI3Ll_cds.2 107 Missense 174- -174 L>I consequence CHI3Ll_cds.3 108 Missense 174- -174 L>I consequence CHI3Ll_cds.4 109 3'

Allele GB:Y08378_1 114 506 506 A>G source isSNP SNP00005491 consequence CHI3Ll_cds.l 106 Intron consequence CHI3Ll_cds.2 107 Intron consequence CHI3Ll_cds.3 108 Intron consequence CHI3Ll_cds.4 109 3'

Allele GB:Y08378_1 114 535 535 A>G source isSNP SNP00005492 consequence CHI3Ll_cds.l 106 Intron consequence CHI3Ll_cds.2 107 Intron consequence CHI3Ll_cds.3 108 Intron consequence CHI3Ll_cds.4 109 3'

Allele GB:Y08378_1 114 641 641 A>G source isSNP SNP00028111 consequence CHI3Ll_cds.l 106 Intron consequence CHI3Ll_cds.2 107 Intron consequence CHI3Ll_cds.3 108 Intron consequence CHI3Ll_cds.4 109 3'

Allele GB:Y08378_1 114 1560 1560 A>G source isSNP SNP00028112 consequence CHI3Ll_cds.l 106 Intron consequence CHI3L1 eds.2 107 Intron TABLE 1 (Cont.) consequence CHI3Ll_cds.3 108 Intron consequence CHI3Ll_cds.4 109 3'

Allele GB:Y08378_1 114 2163 2163 A>G source isSNP SNP00052666 consequence CHI3Ll_cds.l 106 3' consequence CHI3Ll_cds.2 107 Silent 338-338 consequence CHI3Ll_cds.3 108 3' consequence CHI3Ll_cds.4 109 3'

Allele GB:Y08378_1 114 2205 2205 A>G source isSNP SNP00072805 consequence CHI3 l_cds.l 106 3' consequence CHI3Ll_cds.2 107 Silent 352-352 consequence CHI3Ll_cds.3 108 3' consequence CHI3Ll_cds .4 109 3'

Allele GB:Y08378_1 114 2602 2602 A>G source isSNP SNP00076686 consequence CHI3Ll_cds.l 106 3' consequence CHI3Ll_cds.2 107 3' consequence CHI3Ll_cds .3 108 3' consequence CHI3Ll_cds.4 109 3'

GIF CHI3Ll-genomic-fwd .gif

CHI3L2

Full name : chitinase 3-like 2

Link : CHI3L2_link_cdna

Subsequence GB:HSU58514 1 1434 #117 CDS GB:HSU58514.1 1173 bp #118

ORF 37 1209

Allele GB:HSU58514 117 412 412 A>G source isSNP SNP00021152 consequence GB:HSU58514.1 118 Missense 126-126 N>D

Allele GB:HSU58514 117 581 581 A>G source isSNP SNP00021153 consequence GB:HSU58514.1 118 Missense 182-182 A>V

Allele GB:HSU58514 117 972 972 A>G source isSNP SNP00115597 consequence GB:HSU58514.1 118 Silent 312-312 K

Allele GB:HSU58514 117 1204 1204 A>G isSNP SNP00068229 consequence GB:HSU58514.1 118 Silent 390-390 GIF CHI3L2-cdna-fwd.gif Link : CHI3L2_alt_link_cdna Subsequence GB:U58515_1 1500 #119

CDS GB:U58515_1.1 1275 bp #120

ORF 1 1275 Allele GB:U58515_1 119 478 478 A>G source isSNP SNP00021152 consequence GB:U58515_1.1 120 Missense 160-160 N>D

Allele GB:U58515_1 119 647 647 A>G source isSNP SNP00021153 consequence GB:U58515_1.1 120 Missense 216-216 A>V

Allele GB:U58515_1 119 1038 1038 A>G source isSNP SNP001155.97 TABLE 1 (Cont.) consequence GB:U58515_1.1 120 Silent 346-346 K Allele GB:U58515_1 119 1270 1270 A>G source isSNP SNP00068229 consequence GB:U58515_1.1 120 Silent 424-424 GIF CHI3L2-cdna-fwd.gif

CILP

Full name : cartilage intermediate layer protein

Link : CILP_link_cdna

Subsequence GB:AF035408 1 4175 #121

CDS GB:AF035408.1 3555 bp #122

ORF 130 3684 Allele GB:AF035408 121 430 430 A>G source isSNP SNP00123071 consequence GB:AF035408.1 122 Missense 101-101 P>S

Allele GB:AF035408 121 1677 1677 A>G source isSNP SNP00123072 consequence GB:AF035408.1 122 Silent 516-516 R

Allele GB:AF035408 121 3066 3066 A>G source isSNP SNP00020276 consequence GB:AF035408.1 122 Silent 979-979 R

Allele GB:AF035408 121 3263 3263 A>G source isSNP SNP00123073 consequence GB:AF035408.1 122 Missense 1045-1045 Y>C

Allele GB:AF035408 121 3625 3625 A>G source isSNP SNP00055164 consequence GB:AF035408.1 122 Missense 1166-1166 S>G

GIF CILP-cdna-fwd.gif Link : CILP_link_genomic

S Suubbsseeqquueenncc 3ee CILP_cds.l 3606 16639 #123

S Suubbsseeqquueennccee GB:AB022430_1 1 19486 #124

S Suubbsseeqquueenncc :ee CILP_mrna_build.1 1911 17130 #125

C CDDSS CCIILLPP__ceddss..l1 3555 bp 8 exons #123 eexxoonn 3 3660066 3666 exon 5599 5691 exon 6312 6581 exon 7897 8076 exon 8781 9095 exon 9893 10001 exon 11336 11493 exon 14271 16639 mRNA CILP_mrna_build.l 4175 bp 9 exons #125 exon 1911 1933 exon 3500 3666 exon 5599 5691 exon 6312 6581 exon 7897 8076 exon 8781 9095 exon 9893 10001 exon 11336 11493 exon 14271 17130

Allele GB:AB022430_1 124 3 3556677 3567 G>T source wetSNP G GBB:-AABB022430_l.v3567.A>C TABLE 1 (Cont.) consequence CILP_cds .1 123 5'

Allele GB:AB022430_ .1 124 6458 6458 A>G source isSNP SNP00123071 consequence CILP_σds .1 123 Missense 101-101 P>S

Allele GB:AB022430_ .1 124 9874 9874 A>G source wetSNP GB:AB022430_l.v9874. T consequence CILP_cds.l 123 Intron

Allele GB:AB022430_ .1 124 9881 9881 A>G source wetSNP GB:AB022430_l.v9881.OT consequence CILP_cds .1 123 Intron

Allele GB:AB022430_ .1 124 11286 11286 A>T source wetSNP GB:AB022430_1.vll286.T>A consequence CILP_cds .1 123 Intron

Allele GB:AB022430_ .1 124 11491 11491 A>G source wetSNP GB:AB022430_l.vll491.OT consequence CILP_cds .1 123 Missense 395-395 ■ T>I

Allele GB:AB022430_ .1 124 14421 14421 OG source wetSNP GB:AB022430_l.vl4421.G>C consequence CILP_cds .1 123 Missense 446-446 R>T

Allele GB:AB022430_ .1 124 14542 14542 A>G source wetSNP GB:AB022430_l.vl4542.G>A consequence CILP_cds .1 123 Silent 486-486 T

Allele GB:AB022430_ .1 124 ■ 14632 14632 A>G source isSNP SNP00123072 consequence CILP_cds .1 123 Silent 516-516

Allele GB:AB022430. .1 124 15116 15116 A>G source wetSNP GB:AB022430_l.vl5116. A consequence CILP_cds .1 123 Missense 678-678 V>M

Allele GB:AB022430_ _1 124 15670 15670 A>G source wetSNP GB:AB022430_l.vl5670.G>A consequence CILP_cds .1 123 Silent 862-862

Allele GB:AB022430. .1 124 16021 16021 A>G source isSNP SNP00020276 consequence CILP_cds .1 123 Silent 979-979

Allele GB:AB022430_ _1 124 16218 16218 A>G source isSNP SNP00123073 consequence CILP_cds .1 123 Missense 1045-1045 Y>C

Allele GB:AB022430_ .1 124 16580 16580 A>G source isSNP SNP00055164 source wetSNP GB:AB022430_l.vl6580.A>G consequence CILP_cds .1 123 Missense 1166-1166 S>G

GIF CILP-genomic-fwd . gif

COL10A1

Full name : collagen, type X, alpha 1

Link : COL10Al_link_σdna

Subsequence GB:X60382_1 1 3226 #126

CDS GB:X60382_1.2 2043 bp #127

ORF 16 2058

Allele GB:X60382_1 126 95 95 A>G source isSNP SNP00034488 consequence GB:X60382_1.2 127 Missense 27-27 T>M

Allele GB:X60382_1 126 2294 2294 OT TABLE 1 (Cont.) source isSNP SNP00113056 consequence GB :X60382_1.2 127 GIF COL10Al-cdna-fwd . gif

COL11A2

Full name : collagen, type XI, alpha

Link : FL_3421462_link_genomic

Subsequence GB:AL031228_1 1 175737 #128 Subsequence COLllA2_cds.l 93988 122550 #129 Subsequence COLllA2_cds.2 93988 122550 #130 Subsequence COLllA2_cds.3 93988 122550 #131 Subsequence COLllA2_cds.4 93988 122550 #132 Subsequence COLllA2_cds.5 93988 122550 #133 Subsequence COLllA2_cds .6 93988 122550 #134 Subsequence COLllA2_cds.7 93988 122550 #135 Subsequence COLllA2_cds.8 93988 122550 #136 Subsequence COLI1A2_mrna_bui1d 1 93988 122834 #137 Subsequence COLllA2_mrna_build.2 2 93988 122834 #138 Subsequence GB:AL031228_1.20 93762 123536 #139 Subsequence GB:AL031228_1.21 93988 122550 #140 Subsequence COLllA2_mrna_build 3 93769 125002 #141 mRNA GB:AL031228_1.20 6423 bp P 66 exons #139 exon 93762 94069 exon 96759 96908 exon 97040 97250 exon 97704 97866 exon 99410 99601 exon 100450 100527 exon 101174 101236 exon 101904 102083 exon 105058 105117 exon 105223 105264 exon 105498 105560 exon 105896 105970 exon 106423 106509 exon 106741 106797 exon 106944 106997 exon 107102 107155 exon 107255 107308 exon 107496 107549 exon 107740 107793 exon 107876 107920 exon 108043 108096 exon 108522 108566 exon 108763 108816 exon 109003 109047 exon 109183 109236 exon 109463 109507 exon 109742 109795 exon 109925 109969 exon 110159 110212 exon 110547 110654 exon 111648 111701 TABLE 1 (Cont.) exon 112010 112063 exon 112173 112217 exon 112302 112355 exon 112483 112527 exon 112673 112726 exon 112827 112880 exon 113115 113168 exon 113591 113698 exon 113850 113939 exon 114125 114178 exon 114408 114515 exon 114654 114761 exon 114904 114957 exon 115061 115114 exon 115311 115418 exon 115618 115671 exon 115849 115902 exon 116128 116181 exon 116344 116397 exon 116738 116845 exon 117220 117273 exon 117469 117522 exon 117656 117709 exon 118376 118429 exon 118695 118802 exon 118911 118964 exon 119105 119158 exon 119401 119508 exon 119662 119715 exon 120022 120057 exon 120244 120297 exon 120412 120679 exon 121264 121376 exon 121755 121961 exon 122410 123536 mRNA C0LllA2_mrna_build.3 6780 bp 66 exons #141 exon 93769 94341 exon 96759 96908 exon 97040 97250 exon 97704 97866 exon 99410 99601 exon 101174 101236 exon 101904 102083 exon 105058 105117 exon 105223 105264 exon 105498 105560 exon 105896 105970 exon 106423 106509 exon 106741 106797 exon 106944 106997 exon 107102 107155 exon 107255 107308 exon 107496 107549 exon 107740 107793 exon 107876 107920 TABLE 1 (Cont.) exon 108043 108096 exon 108522 108566 exon 108763 108816 exon 109003 109047 exon 109183 109236 exon 109463 109507 exon 109742 109795 exon 109925 109969 exon 110159 110212 exon 110547 110654 exon 111648 111701 exon 112010 112063 exon 112173 112217 exon 112302 112355 exon 112483 112577 exon 112673 112726 exon 112827 112880 exon 113115 113168 exon 113591 113698 exon 113850 113939 exon 114125 114178 exon 114408 114515 exon 114654 114761 exon 114904 114957 exon 115061 115114 exon 115311 115418 exon 115618 115671 exon 115849 115902 exon 116128 116196 exon 116344 116397 exon 116738 116845 exon 117220 117273 exon 117469 117522 exon 117656 117709 exon 118376 118429 exon 118695 118802 exon 118911 118964 exon 119105 119158 exon 119401 119508 exon 120022 120057 exon 120244 120297 exon 120412 120679 exon 121264 121376 exon 121755 121961 exon 122183 122332 exon 122410 123530 exon 124988 125002

CDS COLllA2_cds.6 5157 bp 65 exons #134 exon 93988 94069 exon 96759 96908 exon 97040 97250 exon 97704 97866 exon 99410 99601 exon 100450 100527 exon 101174 101236 TABLE 1 (Cont.) exon 101904 102083 exon 105058 105117 exon 105223 105264 exon 105498 105560 exon 105896 105970 exon 106423 106509 exon 106741 106797 exon 106944 106997 exon 107102 107155 exon 107255 107308 exon 107496 107549 exon 107740 107793 exon 107876 107920 exon 108043 108096 exon 108522 108566 exon 108763 108816 exon 109003 109047 exon 109183 109236 exon 109463 109507 exon 109742 109795 exon 109925 109969 exon 110159 110212 exon 110547 110654 exon 111648 111701 exon 112010 112063 exon 112173 112217 exon 112302 112355 exon 112483 112527 exon 112673 112726 exon 112827 112880 exon 113115 113168 exon 113591 113698 exon 113850 113939 exon 114125 114178 exon 114408 114515 exon 114654 114761 exon 114904 114957 exon 115061 115114 exon 115311 115418 exon 115618 115671 exon 115849 115902 exon 116128 116181 exon 116344 116397 exon 116738 116845 exon 117220 117273 exon 117469 117522 exon 117656 117709 exon 118376 118429 exon 118695 118802 exon 118911 118964 exon 119105 119158 exon 119401 119508 exon 120022 120057 exon 120244 120297 exon 120412 120679 TABLE 1 (Cont.) exon 121264 121376 exon 121755 121961 exon 122410 122550

CDS GB:AL031228_1.21 5211 bp 66 exons #140 exon 93988 94069 exon 96759 96908 exon 97040 97250 exon 97704 97866 exon 99410 99601 exon 100450 100527 exon 101174 101236 exon 101904 102083 exon 105058 105117 exon 105223 105264 exon 105498 105560 exon 105896 105970 exon 106423 106509 exon 106741 106797 exon 106944 106997 exon 107102 107155 exon 107255 107308 exon 107496 107549 exon 107740 107793 exon 107876 107920 exon 108043 108096 exon 108522 108566 exon 108763 108816 exon 109003 109047 exon 109183 109236 exon 109463 109507- exon 109742 109795 exon 109925 109969 exon 110159 110212 exon 110547 110654 exon 111648 111701 exon 112010 112063 exon 112Ϊ73 112217 exon 112302 112355 exon 112483 112527 exon 112673 112726 exon 112827 112880 exon 113115 113168 exon 113591 113698 exon 113850 113939 exon 114125 114178 exon 114408 114515 exon 114654 114761 exon 114904 114957 exon 115061 115114 exon 115311 115418 exon 115618 115671 exon 115849 115902 exon 116128 116181 exon 116344 116397 exon 116738 116845 TABLE 1 (Cont.) exon 117220 117273 exon 117469 117522 exon 117656 117709 exon 118376 118429 exon 118695 118802 exon 118911 118964 exon 119105 119158 exon 119401 119508 exon 119662 119715 exon 120022 120057 exon 120244 120297 exon 120412 120679 exon 121264 121376 exon 121755 121961 exon 122410 122550

CDS COLllA2_σds.7 5049 bp 64 exons #135 exon 93988 94069 exon 96759 96908 exon 97040 97250 exon 97704 97866 exon 99410 99601 exon 101174 101236 exon 105058 105117 exon 105223 105264 exon 105498 105560 exon 105896 105970 exon 106423 106509 exon 106741 106797 exon 106944 106997 exon 107102 107155 exon 107255 107308 exon 107496 107549 exon 107740 107793 exon 107876 107920 exon 108043 108096 exon 108522 108566 exon 108763 108816 exon 109003 109047 exon 109183 109236 exon 109463 109507 exon 109742 109795 exon 109925 109969 exon 110159 110212 exon 110547 110654 exon 111648 111701 exon 112010 112063 exon 112173 112217 exon 112302 112355 exon 112483 112527 exon 112673 112726 exon 112827 112880 exon 113115 113168 exon 113591 113698 exon 113850 113939 exon 114125 114178 TABLE 1 (Cont.) exon 114408 114515 exon 114654 114761 exon 114904 114957 exon 115061 115114 exon 115311 115418 exon 115618 115671 exon 115849 115902 exon 116128 116181 exon 116344 116397 exon 116738 116845 exon 117220 117273 exon 117469 117522 exon 117656 117709 exon 118376 118429 exon 118695 118802 exon 118911 118964 exon 119105 119158 exon 119401 119508 exon 120022 120057 exon 120244 120297 exon 120412 120679 exon 121264 121376 exon 121755 121961 exon 122183 122332 exon 122410 122550

CDS COLllA2_cds.8 4986 bp 63 exons #136 exon 93988 94069 exon 96759 96908 exon 97040 97250 exon 97704 97866 exon 99410 99601 exon 105058 105117 exon 105223 105264 exon 105498 105560 exon 105896 105970 exon 106423 106509 exon 106741 106797 exon 106944 106997 exon 107102 107155 exon 107255 107308 exon 107496 107549 exon 107740 107793 exon 107876 107920 exon 108043 108096 exon 108522 108566 exon 108763 108816 exon 109003 109047 exon 109183 109236 exon 109463 109507 exon 109742 109795 exon 109925 109969 exon 110159 110212 exon 110547 110654 exon 111648 111701 exon 112010 112063 TABLE 1 (Cont.) exon 112173 112217 exon 112302 112355 exon 112483 112527 exon 112673 112726 exon 112827 112880 exon 113115 113168 exon 113591 113698 exon 113850 113939 exon 114125 114178 exon 114408 114515 exon 114654 114761 exon 114904 114957 exon 115061 115114 exon 115311 115418 exon 115618 115671 exon 115849 115902 exon 116128 116181 exon 116344 116397 exon 116738 116845 exon 117220 117273 exon 117469 117522 exon 117656 117709 exon 118376 118429 exon 118695 118802 exon 118911 118964 exon 119105 119158 exon 119401 119508 exon 120022 120057 exon 120244 120297 exon 120412 120679 exon 121264 121376 exon 121755 121961 exon 122183 122332 exon 122410 122550

CDS C0LllA2_cds.l 4890 bp 63 exons #129 exon 93988 94069 exon 96759 96908 exon 97040 97250 exon 97704 97866 exon 99410 99601 exon 105058 105117 exon 105223 105264 exon 105498 105560 exon 105896 105970 exon 106423 106509 exon 106741 106797 exon 106944 106997 exon 107102 107155 exon 107255 107308 exon 107496 107549 exon 107740 107793 exon 107876 107920 exon 108043 108096 exon 108522 108566 exon 108763 108816 TABLE 1 (Cont.) exon 109003 109047 exon 109183 109236 exon 109463 109507 exon 109742 109795 exon 109925 109969 exon 110159 110212 exon 110547 110654 exon 111648 111701 exon 112010 112063 exon 112173 112217 exon 112302 112355 exon 112483 112527 exon 112673 112726 exon 112827 112880 exon 113115 113168 exon 113591 113698 exon 113850 113939 exon 114125 114178 exon 114408 114515 exon 114654 114761 exon 114904 114957 exon 115061 115114 exon 115311 115418 exon 115618 115671 exon 115849 115902 exon 116128 116181 exon 116344 116397 exon 116738 116845 exon 117220 117273 exon 117469 117522 exon 117656 117709 exon 118376 118429 exon 118695 118802 exon 118911 118964 exon 119105 119158 exon 119401 119508 exon 119662 119715 exon 120022 120057 exon 120244 120297 exon 120412 120679 exon 121264 121376 exon 121755 121961 exon 122410 122550

CDS COLllA2_cds.2 4953 bp 64 exons #130 exon 93988 94069 exon 96759 96908 exon 97040 97250 exon 97704 97866 exon 99410 99601 exon 101174 101236 exon 105058 105117 exon 105223 105264 exon 105498 105560 exon 105896 105970 exon 106423 106509 TABLE 1 (Cont.) exon 106741 106797 exon 106944 106997 exon 107102 107155 exon 107255 107308 exon 107496 107549 exon 107740 107793 exon 107876 107920 exon 108043 108096 exon 108522 108566 exon 108763 108816 exon 109003 109047 exon 109183 109236 exon 109463 109507 exon 109742 109795 exon 109925 109969 exon 110159 110212 exon 110547 110654 exon 111648 111701 exon 112010 112063 exon 112173 112217 exon 112302 112355 exon 112483 112527 exon 112673 112726 exon 112827 112880 exon 113115 113168 exon 113591 113698 exon 113850 113939 exon 114125 114178 exon 114408 114515 exon 114654 114761 exon 114904 114957 exon 115061 115114 exon 115311 115418 exon 115618 115671 exon 115849 115902 exon 116128 116181 exon 116344 116397 exon 116738 116845 exon 117220 117273 exon 117469 117522 exon 117656 117709 exon 118376 118429 exon 118695 118802 exon 118911 118964 exon 119105 119158 exon 119401 119508 exon 119662 119715 exon 120022 120057 exon 120244 120297 exon 120412 120679 exon 121264 121376 exon 121755 121961 exon 122410 122550

CDS COLIlA2_cds .3 5307 bp 66 exons #131 exon 93988 94069 TABLE 1 (Cont.) exon 96759 96908 exon 97040 97250 exon 97704 97866 exon 99410 99601 exon 100450 100527 exon 101174 101236 exon 101904 102083 exon 105058 105117 exon 105223 105264 exon 105498 105560 exon 105896 105970 exon 106423 106509 exon 106741 106797 exon 106944 106997 exon 107102 107155 exon 107255 107308 exon 107496 107549 exon 107740 107793 exon 107876 107920 exon 108043 108096 exon 108522 108566 exon 108763 108816 exon 109003 109047 exon 109183 109236 exon 109463 109507 exon 109742 109795 exon 109925 109969 exon 110159 110212 exon 110547 110654 exon 111648 111701 exon 112010 112063 exon 112173 112217 exon 112302 112355 exon 112483 112527 exon 112673 112726 exon 112827 112880 exon 113115 113168 exon 113591 113698 exon 113850 113939 exon 114125 114178 exon 114408 114515 exon 114654 114761 exon 114904 114957 exon 115061 115114 exon 115311 115418 exon 115618 115671 exon 115849 115902 exon 116128 116181 exon 116344 116397 exon 116738 116845 exon 117220 117273 exon 117469 117522 exon 117656 117709 exon 118376 118429 exon 118695 118802 TABLE 1 (Cont.) exon 118911 118964 exon 119105 119158 exon 119401 119508 exon 120022 120057 exon 120244 120297 exon 120412 120679 exon 121264 121376 exon 121755 121961 exon 122183 122332 exon 122410 122550 mRNA _L COLll 2_mrnai_build.1 5174 bp 63 exons #137 exon 93988 94069 exon 96759 96908 exon 97040 97250 exon 97704 97866 exon 99410 99601 exon 105058 105117 exon 105223 105264 exon 105498 105560 exon 105896 105970 exon 106423 106509 exon 106741 106797 exon 106944 106997 exon 107102 107155 exon 107255 107308 exon 107496 107549 exon 107740 107793 exon 107876 107920 exon 108043 108096 exon 108522 108566 exon 108763 108816 exon 109003 109047 exon 109183 109236 exon 109463 109507 exon 109742 109795 exon 109925 109969 exon 110159 110212 exon 110547 110654 exon 111648 111701 exon 112010 112063 exon 112173 112217 exon 112302 112355 exon 112483 112527 exon 112673 112726 exon 112827 112880 exon 113115 113168 exon 113591 113698 exon 113850 113939 exon 114125 114178 exon 114408 114515 exon 114654 114761 exon 114904 114957 exon 115061 115114 exon 115311 115418 exon 115618 115671 TABLE 1 (Cont.) exon 115849 115902 exon 116128 116181 exon 116344 116397 exon 116738 116845 exon 117220 117273 exon 117469 117522 exon 117656 117709 exon 118376 118429 exon 118695 118802 exon 118911 118964 exon 119105 119158 exon 119401 119508 exon 119662 119715 exon 120022 120057 exon 120244 120297 exon 120412 120679 exon 121264 121376 exon 121755 121961 exon 122410 122834

CDS COLllA2_cds.4 4836 bp 62 exons #132 exon 93988 94069 exon 96759 96908 exon 97040 97250 exon 97704 97866 exon 99410 99601 exon 105058 105117 exon 105223 105264 exon 105498 105560 exon 105896 105970 exon 106423 106509 exon 106741 106797 exon 106944 106997 exo^'n 107102 107155 exon 107255 107308 exon 107496 107549 exon 107740 107793 exon 107876 107920 exon 108043 108096 exon 108522 108566 exon 108763 108816 exon 109003 109047 exon 109183 109236 exon 109463 109507 exon 109742 109795 exon 109925 109969 exon 110159 110212 exon 110547 110654 exon 111648 111701 exon 112010 112063 exon 112173 112217 exon 112302 112355 exon 112483 112527 exon 112673 112726 exon 112827 112880 exon 113115 113168 TABLE 1 (Cont.) exon 113591 113698 exon 113850 113939 exon 114125 114178 exon 114408 114515 exon 114654 114761 exon 114904 114957 exon 115061 115114 exon 115311 115418 exon 115618 115671 exon 115849 115902 exon 116128 116181 exon 116344 116397 exon 116738 116845 exon 117220 117273 exon 117469 117522 exon 117656 117709 exon 118376 118429 exon 118695 118802 exon 118911 118964 exon 119105 119158 exon 119401 119508 exon 120022 120057 exon 120244 120297 exon 120412 120679 exon 121264 121376 exon 121755 121961 exon 122410 122550 mRNA COLllA2_rnrna_build.2 5237 bp 64 exons #138 exon 93988 94069 exon 96759 96908 exon 97040 97250 exon 97704 97866 exon 99410 99601 exon 101174 101236 exon 105058 105117 exon 105223 105264 exon 105498 105560 exon 105896 105970 exon 106423 106509 exon 106741 106797 exon 106944 106997 exon 107102 107155 exon 107255 107308 exon 107496 107549 exon 107740 107793 exon 107876 107920 exon 108043 108096 exon 108522 108566 exon 108763 ^' 108816 exon 109003 109047 exon 109183 109236 exon 109463 109507 exon 109742 109795 exon 109925 109969 exon 110159 110212 181 TABLE 1 (Cont.) exon 110547 110654 exon 111648 111701 exon 112010 112063 exon 112173 112217 exon 112302 112355 exon 112483 112527 exon 112673 112726 exon 112827 112880 exon 113115 113168 exon 113591 113698 exon 113850 113939 exon 114125 114178 exon 114408 114515 exon 114654 114761 exon 114904 114957 exon 115061 115114 exon 115311 115418 exon 115618 115671 exon 115849 115902 exon 116128 116181 exon 116344 116397 exon 116738 116845 exon 117220 117273 exon 117469 117522 exon 117656 117709 exon 118376 118429 exon 118695 118802 exon 118911 118964 exon 119105 119158 exon 119401 119508 exon 119662 119715 exon 120022 120057 exon 120244 120297 exon 120412 120679 exon 121264 121376 exon 121755 121961 exon 122410 122834

CDS COLllA2_cds.5 4899 bp 63 exons #133 exon 93988 94069 exon 96759 96908 exon 97040 97250 exon 97704 97866 exon 99410 99601 exon 101174 101236 exon 105058 105117 exon 105223 105264 exon 105498 105560 exon 105896 105970 exon 106423 106509 exon 106741 106797 exon 106944 106997 exon 107102 107155 exon 107255 107308 exon 107496 107549 exon 107740 107793 TABLE 1 (Cont.) exon 107876 107920 exon 108043 108096 exon 108522 108566 exon 108763 108816 exon 109003 109047 exon 109183 109236 exon 109463 109507 exon 109742 109795 exon 109925 109969 exon 110159 110212 exon 110547 110654 exon 111648 111701 exon 112010 112063 exon 112173 112217 exon 112302 112355 exon 112483 112527 exon 112673 112726 exon 112827 112880 exon 113115 113168 exon 113591 113698 exon 113850 113939 exon 114125 114178 exon 114408 114515 exon 114654 114761 exon 114904 114957 exon 115061 115114 exon 115311 115418 exon 115618 115671 exon 115849 115902 exon 116128 116181 exon 116344 116397 exon 116738 116845 exon 117220 117273 exon 117469 117522 exon 117656 117709 exon 118376 118429 exon 118695 118802 exon 118911 118964 exon 119105 119158 exon 119401 119508 exon 120022 120057 exon 120244 120297 exon 120412 120679 exon 121264 121376 exon 121755 121961 exon 122410 122550 ele GB:AL031228. _1 128 122970 1 122970 A>G source isSNP SNP00027609 consequence COLllA2_cds. .6 134 3 consequence GB-.AL031228. .1.21 140 3 consequence C0LllA2_cds. .7 135 3 consequence COLllA2_cds. .8 136 3 consequence C0LllA2_cds, .1 129 3 consequence COLllA2_cds. .2 130 3 consequence COLllA2_cds, .3 131 3 TABLE 1 (Cont.) consequence COLllA2_cds .4 132 3^; consequence COLllA2_cds .5 133 3' GIF COLllA2-genomic-fwd.gif

COL9A2

Full name : collagen, type IX, alpha

Link : FL_3482334_link_cdna

Subsequence FN:3482334CB1 2864 #142 CDS FN:3482334CB1.1 2079 bp #143

ORF 99 2177 Allele FN:3482334CB1 142 1087 1087 A>G source isSNP SNP00032502 consequence FN:3482334CB1.1 143 Missense 330- -330 Q>R

Allele FN:3482334CB1 142 1113 1113 OG source isSNP SNP00107342 consequence FN:3482334CB1.1 143' Missense 339- -339 L>V

Allele FN:3482334CB1 142 1301 1301 A>G source isSNP SNP00107343 consequence FN:3482334CB1.1 143 Silent 401- -401

Allele FN:3482334CB1 142 1345 1345 OG source isSNP SNP00107344 consequence FN:3482334CB1.1 143 Missense 416- -416 G>A

Allele FN:3482334CB1 142 2211 2211 A>G source isSNP SNP00067542 consequence FN-.3482334CB1.1 143 3'

Allele FN:3482334CB1 142 2317 2317 A>G source isSNP SNP00032503 consequence FN:3482334CB1.1 143 GIF COL9A2-cdna-fwd.gif Link : FL_1651412_link_cdna

Subsequence FN: 1651412CB1 1 2869 #144 CDS FN.1651412CB1.1 2067 bp #145

ORF 68 2134 Allele FN:1651412CB1 144 1044 1044 A>G source isSNP SNP00032502 consequence FN: 1651412CB1.1 145 Missense 326- -326 R>Q

Allele FN:1651412CB1 144 1070 1070 OG source isSNP SNP00107342 consequence FN: 1651412CB1.1 145 Missense 335- -335 L>V

Allele FN:1651412CB1 144 1258 1258 A>G source isSNP SNP00107343 consequence FN: 1651412CB1.1 145 Silent 397- -397

Allele FN:1651412CB1 144 1302 1302 OG source isSNP SNP00107344 consequence FN: 1651412CB1.1 145 Missense 412- -412 G>A

Allele FN:1651412CB1 144 2168 2168 A>G source isSNP SNP00067542 consequence FN: 1651412CB1.1 145 3'

Allele FN:1651412CB1 144 2274 2274 A>G source isSNP SNP00032503 consequence FN: 1651412CB1.1 145

GIF COL9A2-cdna-fwd.gif

Link : FL_1651412_link_genomic TABLE 1 (Cont.)

Subsequence GB:AF019406 1 17606 #146

Subsequence GB:AF019406_L651412CD1 1115 17091 #147

Subsequence GB:AF019406_3482334CD1 1115 17091 #148

Subsequence FL_1651412_mrna_build.1 1048 17606 #149

Subsequence FL_3482334_mrna_b ild.l 1017 17606 #150 mRNA FL_1651412_mrna_b ild.l 2649 bp 32 exons #149 exon 1048 1189 exon 2635 2709 exon 3905 3940 exon 4025 4087 exon 5507 5560 exon 5682 5717 exon 5811 5834 exon 6178 6231 exon 6573 6626 exon 6741 6788 exon 7002 7058 exon 7142 7195 exon 7521 7574 exon 7971 8024 exon 8124 8177 exon 8297 8350 exon 10041 10094 exon 10530 10583 exon 10787 10840 exon 12101 12145 exon 12519 12572 exon 13436 13489 exon 13754 13807 exon 13892 13963 exon 14184 14219 exon 14311 14355 exon 14440 14472 exon 14603 14749 exon 15093 15147 exon 15467 15655 exon 16387 16464 exon 16895 17606

CDS GB:AF019406_ 3482334CD1 2079 bp 32 exons #148 exon 1115 1189 exon 2635 2709 exon 3905 3940 exon 4025 4087 exon 5507 5560 exon 5682 5717 exon 5811 5834 exon 6178 6231 exon 6573 6626 exon 6741 6800 exon 7002 7058 exon 7142 7195 exon 7521 7574 exon 7971 8024 exon 8124 8177 exon 8297 8350 TABLE 1 (Cont.) exon 10041 10094 exon 10530 10583 exon 10787 10840 exon 12101 12145 exon 12519 12572 exon 13436 13489 exon 13754 13807 exon 13892 13963 exon 14184 14219 exon 14311 14355 exon 14440 14472 exon 14603 14749 exon 15093 15147 exon 15467 15655 exon 16387 16464 exon 16895 17091 mRNA FL_3482334_mrna_build.l 2692 bp 3 322 exons #150 exon 1017 1189 exon 2635 2709 exon 3905 3940 exon 4025 4087 exon 5507 5560 exon 5682 5717 exon 5811 5834 exon 6178 6231 exon 6573 6626 exon 6741 6800 exon 7002 7058 exon 7142 7195 exon 7521 7574 exon 7971 8024 exon 8124 8177 exon 8297 8350 exon 10041 10094 exon 10530 10583 exon 10787 10840 exon 12101 12145 exon 12519 12572 exon 13436 13489 exon 13754 13807 exon 13892 13963 exon 14184 14219 exon 14311 14355 exon 14440 14472 exon 14603 14749 exon 15093 15147 exon 15467 15655 exon 16387 16464 exon 16895 17606

CDS GB:AF019406_ 1651412CD1 2067 bp 32 exons #147 exon 1115 1189 exon 2635 2709 exon 3905 3940 exon 4025 4087 exon 5507 5560 TABLE 1 (Cont.) exon 5682 5717 exon 5811 5834 exon 6178 6231 exon 6573 6626 exon 6741 6788 exon 7002 7058 exon 7142 7195 exon 7521 7574 exon 7971 8024 exon 8124 8177 exon 8297 8350 exon 10041 10094 exon 10530 10583 exon 10787 10840 exon 12101 12145 exon 12519 12572 exon 13436 13489 exon 13754 13807 exon 13892 13963 exon 14184 14219 exon 14311 14355 exon 14440 14472 exon 14603 14749 exon 15093 15147 exon 15467 15655 exon 16387 16464 exon 16895 17091

Allele GB:AF019406 146 10809 10809 A>G source isSNP SNP00032502 consequence GB:AF019406_3482334CD1 148 Missense 330-330

Q>R consequence GB:AF019406_1651412CD1 147 Missense 326-326

Q>R

Allele GB:AF019406 146 13783 13783 A>G source isSNP ΞNP00107343 consequence GB:AF019406_3482334CD1 148 Silent 401-401

G consequence GB:AF019406_1651412CD1 147 Silent 397-397

G

Allele GB:AF019406 146 17229 17229 A>G source isSNP SNP00032503 consequence GB:AF019406_3482334CD1 148 consequence GB:AF019406_1651412CD1 147

GIF C0L9A2-genomic-fwd .gif

COMP

Full name : cartilage oligomeric matrix protein

Link : FL_1901242_link_cdna

Subsequence FN: 1901242CB1 1 2447 #151

CDS FN:1901242CB1.1 2274 bp #152

ORF 23 2296

Allele FN: 1901242CB1 151 1200 1200 A>G source isSNP SNP00017026 TABLE 1 (Cont.) consequence FN: 1901242CB1.1 152 Missense 393- -393 S>L

Allele FN:1901242CB1 151 1319 1319 OG source isSNP SNP00108392 consequence FN: 1901242CB1.1 152 Missense 433- -433 D>H

Allele FN:1901242CB1 151 1335 1335 OG source isSNP SNP00017027 consequence FN: 1901242CB1.1 152 Missense 438- -438 G>A

Allele FN:1901242CB1 151 1777 1777 A>G source isSNP SNP00017029 consequence FN: 1901242CB1.1 152 Silent 585- -585

GIF COMP-c 3dna-fwd.gif

Link : FL_1901242_ 1ink_genomic

Subsequence GB:AC003107 1 46275 #153

Subsequenc :e GB:AC003107_1901242CD1 32077 23724 #154

Subsequence FL_1901242_mrna_build.1 32099 23582 #155

CDS GB:AC003107_ _.1901242CD1 2274 bp 19 exons #154 exon 32077 31999 exon 31743 31658 exon 31421 31370 exon 30922 30750 exon 30105 29968 exon 29721 29647 exon 29558 29400 exon 29322 29218 exon 29127 29020 exon 28458 28299 exon 27459 27341 exon 27100 27048 exon 26955 26774 exon 26660 26482 exon 26355 26307 exon 25901 25705 exon 25172 25000 exon 24002 23863 exon 23770 23724 mRNA FL_1901242_mrna_build.l 2438 ]bp 19 exons #155 exon 32099 31999 exon 31743 31658 exon 31421 31370 exon 30922 30750 exon 30105 29968 exon 29721 29647 exon 29558 29400 exon 29322 29218 exon 29127 29020 exon 28458 28299 exon 27459 27341 exon 27100 27048 exon 26955 26774 exon 26660 26482 exon 26355 26307 exon 25901 25705 exon 25172 25000 exon 24002 23863 exon 23770 23582 TABLE 1 (Cont.)

Allele GB : AC003107 153 25864 25864 A>G source isSNP SNP00017029 consequence GB : AC003107_1901242CD1 154 Silent 585-585

T Allele GB:AC003107 153 27417 27417 A>G source isSNP SNP00017026 consequence GB:AC003107_1901242CD1 154 Missense 393-393

S>L Allele GB:AC003107 153 32082 32082 A>G source isSNP SNP00017025 consequence GB:AC003107_1901242CD1 154

GIF COMP-genomic-rev.gif

CRLF1

Full name : cytokine receptor-like factor 1

Link : CRLFl_link_cdna

Subsequence GB:AF073515_1 1 1804 #156 CDS GB:AF073515_1.1 1269 bp #157

ORF 204 1472 Allele GB:AF073515_1 156 984 984 A>G source isSNP SNP00015261 consequence GB:AF073515_1.1 157 Missense 261-261 P>S GIF CRLFl-cdna-fwd.gif

CRP

Full name : C-reaσtive protein

Link : CRP link cdna

Subsequenc :e GB:X56214_1 1 1631 #158

CDS GB:X56214_1.1 675 bp #159

ORF 90 764

Allele GB:X56214_1 158 447 447 A>G source isSNP SNP00100892 consequence GB:X56214_1.1 159 Missense 120-120 S>P

Allele GB:X56214_1 158 988 988 A>G source isSNP SNP00029575 consequence GB:X56214_1.1 159

Allele GB:X56214_1 158 1010 1010 A>G source isSNP SNP00076237 consequence GB:X56214_1.1 159

Allele GB:X56214_1 158 1146 1146 OG source isSNP SNP00076238 consequence GB:X56214_1.1 159

Allele GB:X56214_1 158 1175 1175 OT source isSNP SNP00100893 consequence GB:X56214_1.1 159

Allele GB:X56214_1 158 1406 1406 A>G source isSNP SNP00100894 consequence GB:X56214_1.1 159

Allele GB:X56214_1 158 1525 1525 A>G source isSNP SNP00100895 consequence GB:X56214_1.1 159 TABLE 1 (Cont.)

GIF CRP-cdna-fwd.gif Link : CRP_link_genomic

Subsequenc& GB:HUMCRPGA 1 2480 #160

Allele GB :HUMCRPGA 160 865 865 A>G source isSNP SNP00100892

Allele GB :HUMCRPGA 160 1404 1404 A>G source isSNP SNP00029575

Allele GB:HUMCRPGA 160 1426 1426 A>G source isSNP SNP00076237

Allele GB:HUMCRPGA 160 1562 1562 OG source isSNP SNP00076238

Allele GB :HUMCRPGA 160 1591 1591 G>T source isSNP SNP00100893

Allele GB:HUMCRPGA 160 1822 1822 A>G source isSNP SNP00100894

Allele GB:HUMCRPGA 160 1941 1941 A>G source isSNP SNP00100895

Allele GB :HUMCRPGA 160 2045 2045 A>G source isSNP SNP00100896

Allele GB:HUMCRPGA 160 2159 2159 A>G source isSNP SNP00100897

Allele GB :HUMCRPGA 160 2260 2260 A>G source isSNP SNP00006286

CRTL1

Full name : cartilage linking protein 1

Link : CRTLl_link_cdna

Subsequence GB:HSU43328 1 1759 #161

CDS GB:HSU43328.1 1065 bp #162

ORF 118 1182 Allele GB:HSU43328 161 801 801 OG source isSNP SNP00020236 consequence GB:HSU43328.1 162 Silent 228-228

Allele GB:HSU43328 161 1454 1454 A>G source isSNP SNP00002295 consequence GB:HSU43328.1 162 GIF CRTLl-cdna-fwd.gif

CTSC

Full name : cathepsin C Link : CTSC_link_cdna Subsequence GB :NM_ .001814 1838 #163 CDS GB:NM_001814.1 1392 bp #164

ORF 34 1425 Allele GB:NM_001814 163 491 491 A>G source isSNP SNP00006579 consequence GB:NM_001814.1 164 Missense 153-153 T>I

Allele GB:NM_001814 163 1206 1206 G>T source isSNP SNP00006580 consequence GB:NM_001814.1 164 Silent 391-391 T

Allele GB:NM_001814 163 1224 1224 A>G TABLE 1 (Cont.) source isSNP SNP00105444 consequence GB:NM 001814.1 164 Silent 397-397 GIF CTSC-cdna-fwd.gif Link : CTSC_link_genomic

Subsequence CTSC_cds.l 150285 106619 #165 Subsequence CTSC_cds.2 150285 106619 #166 Subsequence GB:AC011088_8 1 164991 #167 Subsequence CTSC_mrna_build.l 150318 106206 #168 CDS CTSC_cds.l 1392 bp 7 exons #165 exon 150285 150114 exon 147695 147550 exon 125167 125001 exon 121931 121776 exon 113258 113143 exon 108877 108746 exon 107121 106619 CDS CTSC_cds.2 1260 bp 6 exons #166 exon 150285 150114 exon 147695 147550 exon 125167 125001 exon 121931 121776 exon 113258 113143 exon 107121 106619 mRNA CTSC_mrna_build.l 1838 bp 7 exons #168 exon 150318 150114 exon 147695 147550 exon 125167 125001 exon 121931 121776 exon 113258 113143 exon 108877 108746 exon 107121 106206

Allele GB:AC011088_.8 167 106820 106820 A>G source isSNP SNP00105444 consequence CTSC_cds.l 165 Silent 397-397 F consequence CTSC_cds.2 166 Silent 353-353 F

Allele GB:AC011088_.8 167 106838 106838 G>T source isSNP SNP00006580 consequence CTSC_cds.l 165 Silent 391-391 T consequence CTSC eds.2 166 Silent 347-347 T

Allele GB:AC011088_ 8 167 122438 122438 A>G source dbSNP gnl|dbSNP|ssl078568_allele source dbSNP gnl j dbSNP] ssl088590_allele source dbSNP gnl|dbSNP|ss382670_allele source dbSNP gnl j dbSNP | ss403413_allele consequence CTSC_cds.l 165 Intron consequence CTSC_cds.2 166 Intron

Allele GB:AC011088_ 8 167 124932 124932 A>T source wetSNP GB:AC011088_8.vl24932.A>T consequence CTSC_cds.l 165 Intron consequence CTSC_cds.2 166 Intron

Allele GB:AC011088_ 8 167 125028 125028 A>G source isSNP SNP00006579 source wetSNP GB:AC011088__8.vl25028.A>G consequence CTSC_cds.l 165 Missense 153-153 I>T consequence CTSC_cds.2 166 Missense 153-153 I>T TABLE 1 (Cont.)

Allele GB:AC011088_ . 167 142996 142996 A>G source dbSNP gnl|dbSNP|ssl530135_allele consequence CTSC_cds.l 165 Intron consequence CTSC_cds .2 166 Intron

Allele GB:AC011088_8 167 150261 150261 A>G source wetSNP GB:AC011088 8.vl50261.G>A consequence CTSC_cds .1 165 Missense L>F consequence CTSC_cds .2 166 Missense L>F

Allele GB:AC011088_8 167 150303 150303 A>G source isSNP SNP00067426 consequence CTSC_cds .1 165 1 consequence CTSC_cds .2 166 !

GIF CTSC-genomic-rev.gif

CTSL

Full name : cathepsin L

Link : CTSL_link_genomic

Subsequence CTSL__cds.l 35962 179319 #169 Subsequence GB:AL160279_2 1 186528 #170 Subsequence CTSL_mrna_build.1 34477 179604 #171 Subsequence CTSL_cds .2 35962 179319 #172 mRNA CTSL_mrna build.1 1577 bp 8 exons #171 exon 34477 34756 exon 35952 36087 exon 36385 36507 exon 36608 36754 exon 36943 37167 exon 37931 38093 exon 38739 38856 exon 17922C ) 179604

CDS CTSL_eds .1 1002 bp 7 exons #169 exon 35962 36087 exon 36385 36507 exon 36608 36754 exon 36943 37167 exon 37931 38093 exon 38739 38856 exon 17922C ) 179319

CDS CTSL_cds.2 777 bp 6 exc #172 exon 35962 36087 exon 36385 36507 exon 36608 36754 exon 37931 38093 exon 38739 38856 exon 179220 179319 Allele GB:AL160279_2 170 35919 35919 OG source wetSNP GB :AL160279_2 .v35919 . OG consequence CTSL_cds .1 169 5 ' consequence CTSL_cds .2 172 5 '

Allele GB:AL160279_2 170 36118 36118 A>G source wetSNP GB : AL160279_2 .v36118 . T consequence CTSL_cds .1 169 Intron consequence CTSL_cds .2 172 Intron TABLE 1 (Cont.)

Allele GB:AL160279_2 170 36191 36191 G>T source wetSNP GB:ALl60279_2.v36191.OA consequence CTSL_cds.l 169 Intron consequence CTSL_cds .2 172 Intron

Allele GB: L160279_2 170 44998 44998 A>G source isSNP SNP00043782 consequence CTSL_cds .1 169 Intron consequence CTSL_cds.2 172 Intron

Allele GB:AL160279_2 170 45748 45748 A>G source isSNP SNP00007530 consequence CTSL_cds .1 169 Intron consequence CTSL_cds.2 172 Intron

Allele GB:AL160279_2 170 45833 45833 OG source isSNP SNP00100366 consequence CTSL_cds .1 169 Intron consequence CTSL_cds.2 172 Intron

Allele GB :AL160279_2 170 46188 46188 A>G ^' source isSNP SNP00100365 consequence CTSL_cds .1 169 Intron consequence CTSL_cds.2 172 Intron

Allele GB:AL160279_2 170 46599 46599 OG source isSNP SNP00061067 consequence CTSL_cds .1 169 Intron consequence CTSL_cds.2 172 Intron

Allele GB:AL160279_2 170 46662 46662 OG source isSNP SNP00100364 consequence CTSL_cds .1 169 Intron consequence CTSL_cds.2 172 Intron

Allele GB :AL160279_2 170 65760 65760 A>G source isSNP SNP00048929 consequence CTSL_cds .1 169 Intron consequence CTSL_cds.2 172 Intron

Allele GB:AL160279 2 170 81133 81133 A>G source dbSNP gnl | dbSNP| ss920176_allele source dbSNP gnl j dbSNP j ssl066694_allele source dbSNP gnl j dbSNP | ss402532_allele consequence CTSL_cds.l 169 Intron consequence CTSL_cds .2 172 Intron

Allele GB:AL160279_2 170 104937 104937 A>G source isSNP SNP00055641 consequence CTSL_cds.l 169 Intron consequence CTSL_cds .2 172 Intron

Allele GB:AL160279_2 170 115466 115466 A>G source isSNP SNP00100363 consequence CTSL_cds .1 169 Intron consequence CTSL_cds .2 172 Intron

Allele GB:AL160279_2 170 127655 127655 A>T source dbSNP gnl | dbSNP| ss810769_allele consequence CTSL_cds .1 169 Intron consequence CTSL_cds .2 172 Intron

Allele GB:AL160279_2 170 149731 149731 A>G source dbSNP gnl | dbSNP| ssl452230_allele consequence CTSL_cds .1 169 Intron consequence CTSL_cds .2 172 Intron

GIF CTSL-genomic-fwd.gif TABLE 1 (Cont.)

DAF

Full name : decay accelerating factor for complement

Link : DAF_link_genomic

Subsequence DAF_cds.l 131174 169024 #173

Subsequence DAF_cds.2 131174 169024 #174

Subsequence GB:AC031978_3 1 170170 #175

Subsequence DAF_mrna_build.l 131109 169897 #176

CDS DAF_cds .1 1146 bp 10 exons #173 exon 131174 131273 exon 131790 131975 exon 133967 134158 exon 135030 135129 exon 136160 136245 exon 140516 140704 exon 146101 146226 exon 146737 146817 exon 148808 148828 exon 168960 169024

CDS DAF_cds .2 1125 bp 9 exons #174 exon 131174 131273 exon 131790 131975 exon 133967 134158 exon 135030 135129 exon 136160 136245 exon 140516 140704 exon 146101 146226 exon 146737 146817 exon 168960 169024 mRNA DAF_mr: 2084 bp 10 exons #176 exon 131109 131273 exon 131790 131975 exon 133967 134158 exon 135030 135129 exon 136160 136245 exon 140516 140704 exon 146101 146226 exon 146737 146817 exon 148808 148828 exon 168960 169897

Allele GB:AC0 78_3 175 132041 132041 A>G source wetSNP GB : AC031978_3 .vl32041 . OT consequence DAF_cds .1 173 Intron consequence DAF_cds .2 174 Intron

Allele GB:AC031978_3 175 146352 146352 A>G source isSNP SNP00072272 consequence DAF_cds .1 173 Intron consequence DAF_cds .2 174 Intron

Allele GB:AC031978_3 175 146611 146611 A>G source isSNP SNP00072273 consequence DAF_cds .1 173 Intron consequence DAF_cds .2 174 Intron

Allele GB:AC031978_3 175 146659 146659 A>G source isSNP SNP00030860 TABLE 1 (Cont.) consequence DAF_cds.l 173 Intron consequence DAF_cds.2 174 Intron

Allele GB:AC031978_ 3 175 165604 165604 A>G source isSNP SNP00102533 consequence DAF_cds.l 173 Intron consequence DAF_cds.2 174 Intron

Allele GB:AC031978_3 175 165743 165743 A>G source isSNP SNP00102534 consequence DAF_cds .1 173 Intron consequence DAF_cds .2 174 Intron^' GIF DAF-genomic-fwd.gif

E2F6

Full name : E2F transcription factor 6

Link : E2F6_link_cdna

Subsequence GB:AF041381 1 2027 #177 Allele GB:AF041381 177 1399 1399 A>G source isSNP SNP00002319

EGF

Full name : EGF

Link : EGF_link_σdna

Subsequence GB:HSEGFRER 1 4871 #178 CDS GB :HSEGFRER.1 3624 bp #179

ORF 437 4060 Allele GB:HSEGFRER 178 4453 4453 A>G source isSNP SNP00043643 consequence GB :HSEGFRER.1 179 GIF EGF-cdna-fwd.gif Link : EGF_link_genomic

Subsequence GB:AC005509 1 143391 #180 Subsequence GB:AC004050 270590 143492 #181 Subsequence EGF_cds.l 64892 166730 #182 Subsequence EGF_mrna_build.l 64456 167538 #183 CDS EGF_cds. 3624 bp 24 exons #182 exon 64892 65018 exon 92502 92701 exon 94810 94991 exon 95398 95625 exon 96629 96831 exon 110868 110993 exon 112423 112545 exon 113419 113541 exon 114729 114854 exon 115957 116093 exon 120527 120675 exon 126259 126363 exon 127568 127791 exon 131528 131695 exon 132382 132531 exon 134978 135097 TABLE 1 (Cont.) exon 139300 139416 exon 143859 143984 exon 148522 148644 exon 150008 150155 exon 154954 155121 exon 159780 159897 exon 163427 163505 exon 166477 166730 mRNA EGF_mrna_build.l 4868 bp 24 exons #183 exon 64456 65018 exon 92502 92701 exon 94810 94991 exon 95398 95625 exon 96629 96831 exon 110868 110993 exon 112423 112545 exon 113419 113541 exon 114729 114854 exon 115957 116093 exon 120527 120675 exon 126259 126363 exon 127568 127791 exon 131528 131695 exon 132382 132531 exon 134978 135097 exon 139300 139416 exon 140140 140265 exon 148522 148644 exon 150008 150155 exon 154954 155121 exon 159780 159897 exon 163427 163505 exon 166477 167538

Allele GB:AC005509 180 70903 70903 A>G source dbSNP gnl|dbSNP|ss875266_allele consequence EGF_cds .1 182 Intron

Allele GB:AC005509 180 92638 92638 A>G source wetSNP GB:AC005509.v92638 .OT consequence EGF_cds .1 182 Silent 88-88 I

Allele GB:AC005509 180 92670 92670 A>G source wetSNP GB:AC005509.v92670 .A>G consequence EGF_cds .1 182 Missense 99-99 Q Q>R

Allele GB:AC005509 180 92763 92763 A>G source wetSNP GB:AC005509.v92763 .OT consequence EGF_cds .1 182 Intron

Allele GB:AC005509 180 94933 94933 A>G source wetSNP GB:AC005509.v94933 .OT consequence EGF_cds .1 182 Missense 151-151 H>Y

Allele GB:AC005509 180 95444 95444 OG source wetSNP GB:AC005509.v95444 .G>C consequence EGF_cds .1 182 Missense 186-186 D>H

Allele GB:AC005509 180 96578 96578 G>T source wetSNP GB:AC005509.v96578 .A>C consequence EGF_cds .1 182 Intron

Allele GB:AC005509 180 96660 96660 OG TABLE 1 (Cont.) source wetSNP GB:AC005509.v96660.G>C consequence EGF_cds .1 182 Missense 257-257 D>H

Allele GB:AC005509 180 96842 96842 A>G source wetSNP GB:AC005509.v96842.G>A consequence EGF_cds .1 182 Intron

Allele GB:AC005509 180 96853 96853 A>G source wetSNP GB:AC005509.v96853.G>A consequence EGF_cds .1 182 Intron

Allele GB:AC005509 180 100795 100795 OT source dbSNP gnl|dbSNP|ss48546_allele source dbSNP gnljdbSNP|ss569965_allele consequence EGF_cds.l 182 Intron

Allele GB:AC005509 180 112451 112451 A>G source wetSNP GB:AC005509.vll2451.T>C consequence EGF_cds.l 182 Silent 365-365

Allele GB:AC005509 180 113396 113396 A>G source wetSNP GB:AC005509.vll3396.T>C consequence EGF_cds.l 182 Intron

Allele GB:AC005509 180 113521 113521 A>G source wetSNP GB:AC005509.vll3521.G>A consequence EGF_cds.l 182 Missense 431-431 R>K

Allele GB:AC005509 180 114696 114696 A>G source wetSNP GB:AC005509.vll4696.OT consequence EGF_cds.l 182 Intron

Allele GB:AC005509 180 126323 126323 A>G source wetSNP GB:AC005509.vl26323.A>G consequence EGF_cds.l 182 Missense 597-597 I>V

Allele GB:AC005509 180 127715 127715 A>G source wetSNP GB:AC005509.vl27715.C>T consequence EGF_cds.l 182 Silent 659-659

Allele GB:AC005509 180 131547 131547 A>G source wetSNP GB:AC005509.vl31547.A>G consequence EGF_cds.l 182 Silent 691-691

Allele GB:AC005509 180 131598 131598 A>G source wetSNP GB:AC005509.vl31598.G>A consequence EGF_cds.l 182 Missense 708-708 M>I

Allele GB:AC005509 180 131641 131641 OG source wetSNP GB:AC005509.vl31641.G>C consequence EGF_cds.l 182 Missense 723-723 G>R

Allele GB:AC005509 180 132511 132511 A>T source wetSNP GB:AC005509.vl32511.A>T consequence EGF_cds.l 182 Missense 784-784 D>V

Allele GB:AC005509 180 139281 139281 A>G source wetSNP GB:AC005509.vl39281.G>A consequence EGF_cds.l 182 Intron

Allele GB:AC005509 180 139333 139333 A>G source wetSNP GB:AC005509.vl39333.T>C consequence EGF_cds.l 182 Missense 842-842 M>T

Allele GB:AC004050 181 126737 126737 G>T source wetSNP GB:AC004050.vl26737. A consequence EGF_cds.l 182 Intron

Allele GB:AC004050 181 122948 122948 A>G source isSNP SNP00118827 consequence EGF_cds.l 182 Intron

Allele GB:AC004050 181 122045 122045 A>T TABLE 1 (Cont.) source wetSNP GB:AGO 04050. l22045.A>T consequence EGF_cds.l 182 Missense 920-920 E>V

Allele GB:AC004050 181 110980 110980 G>T source isSNP SNP00101773 consequence EGF_cds.l 182 Intron

Allele GB:AC004050 181 110796 110796 A>G source wetSNP GB:AC004050. ll0796.A>G consequence EGF_cds.l 182 Silent 1063-1063

Allele GB:AC004050 181 104082 104083 GOGCC source wetSNP GB:AC004050.V104082.GOGCC consequence EGF_σds.l 182 Fra eshift 1134-1135

Allele GB:AC004050 181 103468 103468 A>G source isSNP SNP00043643 consequence EGF_cds.l 182

GIF ΞGF- -genomic-fwd.gif

FDFTl

Full name : farnesyl-diphosphate farnesyltransferase 1

Link : FDFTl_link_cdna

Subsequence GB:FDFTl 1 1649 #184 CDS GB: FDFTl.1 1254 bp #185

ORF 45 1298 Allele GB :FDFTl 184 65 65 A>G source isSNP SNP00072434 consequence GB :FDFTl.1 185 Silent 7-7

Allele GB :FDFTl 184 178 178 A>G source isSNP SNP00065489 consequence GB:FDFTl.1 185 Missense 45-45 K>R

Allele GB:FDFTl 184 245 245 A>G source isSNP SNP00018570 consequence GB: FDFTl.1 185 Silent 67-67 N

Allele GB : FDFTl 184 590 590 A>G source isSNP SNP00123116 consequence GB:FDFTl.1 185 Silent 182-182

Allele GB : FDFTl 184 1016 1016 OG source isSNP SNP00003188 consequence GB:FDFTl.1 185 Silent 324-324

Allele GB :FDFTl 184 1220 1220 A>G source isSNP SNP00123117 consequence GB:FDFTl.1 185 Silent 392-392

Allele GB : FDFTl 184 1532 1532 A>G source isSNP SNP00003189 consequence GB :FDFTl.1 185 GIF FDFTl-cdna-fwd.gif Link : FDFTl_link_genomic

Subsequence FDFTl_cds.l 5681 37973 #186 Subsequence GB:AC025857_2_000033 1 19420 #187 Subsequence GB:AC025857_2_000021 19521 25487 #188 Subsequence GB:AC025857_2_000014 29099 25588 #189 Subsequence GB:AC025857_2_000029 29200 40859 #190 Subsequence FDFTl mrna_build.l 5639 38324 #191 mRNA FDFTl_mrna_build.1 1647 bp 8 exons #191 exon 5639 5779 TABLE 1 (Cont.) exon 11642 11739 exon 12515 12698 exon 24238 24366 exon 26209 26400 exon 29608 29784 exon 30882 31034 exon 37752 38324

CDS FDFTl. _cds .1 1254 bp 8 exons #186 exon 5681 5779 exon 11642 11739 exon 12515 12698 exon 24238 24366 exon 26209 26400 exon 29608 29784 exon 30882 31034 exon 37752 37973

Allele GB:AC025857_2. _000033 187 5701 5701 A>G source isSNP SNP00072434 consequence FDFTl_σds.l 186 Silent 7-7 L

Allele GB:AC025857. _2_000033 187 6103 6103 OG source isSNP SNP00072231 consequence FDFTl_cds.l 186 Intron

Allele GB:AC025857. _2_000033 187 11676 11676 A>G source isSNP SNP00065489 consequence FDFTl_cds.l 186 Missense 45-45 K>R

Allele GB:AC025857. _2_000014 189 2856 2856 A>G source isSNP SNP00123116 consequence FDFTl_cds.l 186 Silent 182-182

Allele GB:AC025857. _2_000029 190 1775 1775 OG source isSNP SNP00003188 consequence FDFTl_cds.l 186 Silent 324-324

Allele GB:AC025857. _2_000029 190 5704 5704 A>G source isSNP SNP00096026 consequence FDFTl_cds.l 186 Intron

Allele GB:AC025857. _2_000029 190 8528 8528 A>G source isSNP SNP00105147 consequence FDFTl_cds.l 186 Intron

Allele GB:AC025857. _2_000029 190 8696 8696 A>G source isSNP SNP00123117 consequence FDFTl_cds.l 186 Silent 392-392

Allele GB:AC025857. _2_000029 190 9008 9008 A>G source isSNP SNP00003189 consequence FDFTl_cds.l 186 3'

Allele GB:AC025857. _2_000029 190 9148 9148 OT source isSNP SNP00003190 consequence FDFTl_cds.l 186 3'

GIF FDFTl-genomic-fwd. gif

FGF1

Full name : Fibroblast growth factor 1 (acidic)

Link : FGFl_link_cdna

Subsequence GB:X51943_1 1 2259 #192

CDS GB:X51943_1.1 468 bp #193 TABLE 1 (Cont.)

ORF 35 502 Allele GB:X51943_1 192 590 590 A>G source isSNP SNP00075582 consequence GB:X51943_1.1 193

Allele GB:X51943_1 192 785 785 G>T source isSNP SNP00075583 consequence GB:X51943_1.1 193

Allele GB:X51943_1 192 1855 1855 A>G source isSNP SNP00069845 consequence GB:X51943_1.1 193

Allele GB:X51943_1 192 2007 2007 OG source isSNP SNP00075584 consequence GB:X51943_1.1 193 GIF FGFl-cdna-fwd.gif Link : FL_2535357_link_genomic

Subsequence GB:AC005370 1 76416 #194 Subsequence GB:AC005370_3284782CD1 45026 63860 #195 Subsequence FL_3284782_mrna_build.1 44979 67355 #196 mRNA FL_3284782_mrna_build.l 920 bp 4 exons #196 exon 44979 45194 exon 58348 58451 exon 63669 64259 exon 67347 67355 CDS GB:AC005370_3284782CD1 465 bp 3 exons #195 exon 45026 45194 exon 58348 58451 exon 63669 63860 Allele GB:AC005370 194 63951 63951 A>G source isSNP SNP00075582 consequence GB:AC005370_3284782CD1 195

Allele GB:AC005370 194 64146 64146 G>T source isSNP SNP00075583 consequence GB:AC005370_3284782CD1 195

Allele GB:AC005370 194 65119 65119 G>T source isSNP SNP00012384 consequence GB:AC005370_3284782CD1 195

Allele GB:AC005370 194 65217 65217 A>G source isSNP SNP00069845 consequence GB:AC005370_3284782CD1 195

Allele GB:AC005370 194 65369 65369 OG source isSNP SNP00075584 consequence GB:AC005370_3284782CD1 195

Allele GB:AC005370 194 66005 66005 A>G source isSNP SNP00045433 consequence GB:AC005370_3284782CD1 195

GIF FGFl-genomic-fwd.gif

FGF2

Full name : fibroblast growth factor 2 (basic)

Link : FGF2_link_cdna

Subsequence GB:FGF2 1 6757 #197 CDS GB:FGF2.1 633 bp #198

ORF 302 934 TABLE 1 (Cont.)

Allele GB:FGF2 197 1651 1651 G>T source isSNP SNP00023270 consequence GB:FGF2.1 198 3'

Allele GB : FGF2 197 1691 1691 A>G source isSNP SNP00058183 consequence GB:FGF2.1 198 3'

Allele GB :FGF2 197 4603 4603 A>G source isSNP SNP00036340 consequence GB:FGF2.1 198 3'

Allele GB :FGF2 197 4909 4909 A>G source isSNP SNP00036341 consequence GB:FGF2.1 198 3'

Allele GB :FGF2 197 5455 5455 A>G source isSNP SNP00123025 consequence GB:FGF2.1 198 3'

Allele GB:FGF2 197 5466 5466 OG source isSNP SNP00036342 consequence GB:FGF2.1 198 3'

Allele GB :FGF2 197 5892 5892 G>T source isSNP SNP00062439 consequence GB:FGF2.1 198 3'

Allele GB:FGF2 197 5937 5937 A>G source isSNP SNP00062440 consequence GB:FGF2.1 198 3'

GIF FGF2- -cdna-fwd.gif

FGFR1

Full name : Fibroblast growth factor receptor-1 Link : FGFRl_link_cdna Subsequence GB:M3 185_1 1 3365 #199 CDS GB:M34185_1.1 2202 bp #200

ORF 256 2457 Allele GB:M34185_1 199 1471 1471 A>G source isSNP SNP00107960 consequence GB:M34185_1.1 200 Missense 406-406 A>T

Allele GB:M34185_1 199 3224 3224 G>T source isSNP SNP00107961 consequence GB:M34185_1.1 200 3' GIF FGFRl-cdna-fwd.gif

FMOD

Full name : fibromodulin

Link : FMOD_link_cdna

Subsequenc :e GB:FMOD 1 2863 #20:

CDS GB:FMOD.l 1131 bp #202

ORF 21 1151

Allele GB :FMOD 201 2653 2653 OG source isSNP SNP00001499 consequence : GB:FMOD.1 202 3'

Allele GB : FMOD 201 2739 2739 A>G source isSNP SNP00001500 TABLE 1 (Cont.) consequence GB:FMOD.l 202 GIF FMOD-cdna-fwd.gif

FRZB

Full name : frizzled-related protein

Link : FRZB_link_cdna

Subsequence GB:U91903_1 1 1909 #203

CDS GB:U91903 1.1 978 bp #204

ORF 70 1047 Allele GB:U91903_1 203 667 667 A>G source isSNP SNP00016790 consequence GB:U91903_1.1 204 Missense 200-200 R>W

Allele GB:U91903_1 203 1039 1039 OG source isSNP SNP00001065 consequence GB:U91903_1.1 204 Missense 324-324 R>G

Allele GB:U91903_1 203 1259 1259 A>G source isSNP SNP00001066 consequence GB:U91903_1.1 204 3'

Allele GB:U91903_1 203 1305 1305 A>G source isSNP SNP00016791 consequence GB:U91903_1.1 204 GIF FRZB-cdna-fwd.gif

FST

Full name : Follistatin

Link : FST link cdna

Subsequence GB:FST 1 954 #205

CDS GB:FST.l 954 bp #206

ORF 1 954

Allele GB:FST 205 454 454 A>G source isSNP SNP00015508 consequence GB:FST.l 206 Missense 152-152 E>K

Allele GB : FST 205 853 853 OG source isSNP SNP00052278 consequence GB : FS .1 206 Missense 285-285 A>P

GIF FST-cdna-fwd.gif

Link : FST _1ink_genomic

Subsequence FST_cds .1 77877 73442 #207

Subsequence GB:AC008901_2 1 192639 #208

Subsequence FST_mrna_build.1 77877 73440 #209

CDS F FSSTT__ccddss..ll 9 95511 bbpp 5 exons #207 eexxoonn 7 777887777 7 777779933 exon 75788 75597 exon 75164 74946 exon 74599 74375 exon 73671 73442 mRNA FST_mrna_buiId.1 953 bp exons #209 exon 77877 77793 exon 75788 75597 exon 75164 74946 exon 74599 74375 TABLE 1 (Cont.) exon 73671 73440 Allele GB :AC008901_2 208 73454 73454 A>G source wetSNP GB:AC008901_2.v73454.G>A consequence FST_σds .1 207 Silent 313-313 S Allele GB:AC008901_2 208 73540 73540 OG source isSNP SNP00052278 consequence FST_cds .1 207 Missense 285-285 A>P Allele GB:AC008901_2 208 74988 74988 A>G source isSNP SNP00015508 consequence FST_cds .1 207 Missense 152-152 E>K Allele GB:AC008901_2 208 76361 76361 OG source dbSNP gnl | dbSNP| ss42460_allele consequence FST_cds .1 207 Intron Allele GB:AC008901_2 208 76373 76373 A>G source dbSNP gnl | dbSNP | ssl048607_allele source dbSNP gnl j dbSNP j ss226044_allele consequence FST_cds .1 207 Intron Allele GB: C008901_2 208 76384 76384 A>G source dbSNP gnl | dbSNP| ss839844_allele consequence FST_cds .1 207 Intron GIF FST-genomic-rev.gif

G0S2

Full name : putative lymphocyte G0\/G1 switch gene

Link : FL_3732868_link_genomic

Subsequence GB:HS28O10 1 97700 #210 Subsequence GB:HS28O10_3732868CDl 52369 52680 #211 Subsequence FL_3732868_mrna_build.1 52008 53073 #212 mRNA FL_3732868_mrna_build.l 963 bp 2 exons #212 exon 52008 52233 exon 52337 53073 CDS GB:HS28O10_3732868CDl 312 bp 1 exon #211 exon 52369 52680 Allele GB:HS28O10 210 52341 52341 A>G source isSNP SNP00039143 source wetSNP GB:HS28O10.v52341.T>C consequence GB:HS28O10_3732868CDl 211 5' GIF G0S2-genomic-fwd.gif

GADD34

Full name : growth arrest and DNA-damage-inducible 34

Link : GADD34_link_cdna

Subsequenc :e GB:HSU83981 1 2331 #213

CDS GB.HSU83981.1 2025 bp #214

ORF 223 2247

Allele GB:HSU83981 213 205 205 A>G source isSNP SNP00116263 consequence GB:HSU83981.1 214

Allele GB:HSU83981 213 314 314 A>G source isSNP SNP00116264 consequence GB.HSU83981.1 214 Missense 31-31 R>H TABLE 1 (Cont.)

Allele GB:HSU83981 213 316 316 A>G source isSNP SNP00029694 consequence GB.HSU83981.1 214 Missense 32-32 A>T

Allele GB:HSU83981 213 974 974 C>G source isSNP SNP00006368 consequence GB.HSU83981.1 214 Missense 251-251 R>P

Allele GB:HSU83981 213 1051 1051 A>G source isSNP SNP00006369 consequence GB:HSU83981.1 214 Missense 277-277 >Ξ

Allele GB:HSU83981 213 1156 1156 A>G source isSNP SNP00006370 consequence GB.HSU83981.1 214 Missense 312-312 G>S

Allele GB:HSU83981 213 1605 1605 A>G source isSNP SNP00069978 consequence GB:HSU83981.1 214 Silent 461-461

Allele GB:HSU83981 213 1650 1650 OT source isSNP SNP00069979 consequence GB.HSU83981.1 214 Missense 476-476 R>S

Allele GB:HSU83981 213 2011 2011 A>G source isSNP SNP00006372 consequence GB:HSU83981.1 214 Missense 597-597 T>A

Allele GB:HSU83981 213 2184 2184 A>G source isSNP SNP00006373 consequence GB:HSU83981.1 214 Silent 654-654

Allele GB:HSU83981 213 2199 2199 OG source isSNP SNP00006374 consequence GB.HSU83981.1 214 Silent 659-659 GIF GADD34-cdna-fwd.gif Link : GADD34_link_genomic

Subsequence GADD34_cds.l 221390 224129 #215 Subsequence GB:AC026803_2 1 247509 #216 Subsequence GADD34_mrna_build 1 220595 224213 #217 mRNA GADD34 mrna ouild.1 2331 bp 3 exons #217 exon 220595 220807 exon 221381 223054 exon 223770 224213

CDS GADD34_cds.l 2025 bp 2 exons #215 exon 221390 223054 exon 223770 224129

Allele GB:AC026803_2 216 221481 221481 A>G source isSNP SNP00116264 consequence GADD34_cds.l 215 Missense 31-31 R>H

Allele GB:AC026803_2 216 221483 221483 A>G source isSNP SNP00029694 consequence GADD34_σds.l 215 Missense 32-32 A>T

Allele GB:AC026803_2 216 221941 221941 A>G source wetSNP GB:AC026803_2.v221941.G>A consequence GADD34_cds .1 215 Silent 184-184

Allele GB:AC026803_2 216 221985 221985 A>G source wetSNP GB:AC026803.2.v221985.T>C consequence GADD34_cds .1 215 Missense 199-199 V>A

Allele GB:AC026803_2 216 222141 222141 OG source isSNP SNP00006368 source wetSNP GB:AC026803_2. 222141.G>C consequence GADD34 cds.l 215 Missense 251-251 R>P TABLE 1 (Cont.)

Allele GB:AC026803_2 216 222218 222218 A>G source isSNP SNP00006369 consequence GADD34_cds.l 215 Missense 277-277 K>E Allele GB:AC026803_2 216 222323 222323 A>G source isSNP SNP00006370 consequence OADD34_cds.l 215 Missense 312-312 G>S Allele GB:AC026803_2 216 222772 222772 A>G source isSNP SNP00069978 consequence GADD34_cds.l 215 Silent 461-461 L Allele GB:AC026803_2 216 222817 222817 OT source isSNP SNP00069979 consequence GADD34_cds.l 215 Missense 476-476 R>S Allele GB:AC026803_2 216 223893 223893 A>G source isSNP SNP00006372 consequence GADD34_cds.l 215 Missense 597-597 T>A Allele GB:AC026803_2 216 224066 224066 A>G source isSNP SNP00006373 consequence GADD34_cds.l 215 Silent 654-654 A Allele GB:AC026803_2 216 224081 224081 OG source isSNP SNP00006374 consequence GADD34_cds.l 215 Silent 659-659 S GIF GADD34-genomic-fwd.gif

GLI

Full name : glioma-associated oncogene homolog

Link : GLI_link_cdna

Subsequence GB:NM_005269_1 1 3600 #218 CDS GB:NM_005269_1.1 3321 bp #219

ORF 79 3399 Allele GB:NM_005269_1 218 2179 2179 A>G source isSNP SNP00018615 consequence GB:NM_005269_1.1 2 21199 M Miisssseennssee 701-701 R>G Allele GB:NM_005269_1 218 2202 2202 A>G source isSNP SNP00072776 consequence GB:NM_005269_1.1 2 21199 S Siilleenntt 708-708 E Allele GB:NM_005269_1 218 2876 2876 A>G source isSNP SNP00112595 consequence GB:NM_005269_1.1 2 21199 M Miisssseennssee 933-933 G>D Allele GB:NM_005269_1 218 3243 3243 OG source isSNP SNP00018616 consequence GB:NM__005269_1.1 2 21199 M Miisssseennssee 1055-1055 E>D Allele GB:NM_005269_1 218 3376 3376 OG source isSNP SNP00018617 consequence GB:NM__005269_1.1 2 21199 M Miisssseennssee 1100-1100 E>Q GIF GLI-cdna-fwd.gif

GLI3

Full name : GLI-Kruppel family member GLI3

Link : GLI3_link_cdna

Subsequence GB:NM_000168_1 1 5046 #220 CDS GB:NM_000168_1.1 4791 bp #221 TABLE 1 (Cont.)

ORF 55 4845 Allele GB:NM_000168_1 220 4502 4502 A>G source isSNP SNP00031650 consequence GB:NM_000168_1.1 222211 MMiisssseennssee 11448833--11448833 G>D Allele GB:NM_000168_1 220 4663 4663 A>G source isSNP SNP00073523 consequence GB:NM_000168_1.1 222211 MMiisssseennssee 11553377--11553377 R>C GIF GLI3-cdna-fwd.gif

HAS1

Full name : hyaluronan synthase 1

Link : HASl_link_cdna

Subsequence GB:NM_001523 2088 #222 CDS GB:NM_001523.1 1737 bp #223

ORF 36 1772 Allele GB:NM_001523 222 75 75 A>G source isSNP SNP00096015 consequence GB:NM_001523.1 223 Missense 14-14 R>C

Allele GB:NM_001523 222 1889 1889 G>T source isSNP SNP00064738 consequence GB:NM_001523.1 223 3' GIF HASl-cdna-fwd.gif Link- : HASl_link_genomic

Subsequence HASl_cds.l 153154 142648 #224 Subsequence GB:AC018755_2 1 231222 #225 Subsequence HASl_mrna_build.1 153189 142333 #226 CDS HASl_cds.l 1737 bp 5 exons #224 exon 153154 153146 exon 149119 148427 exon 146414 146189 exon 145609 145477 exon 143323 142648 mRNA HASl_mrna_build.l 2087 bp 5 exons #226 exon 153189 153146 exon 149119 148427 exon 146414 146189 exon 145609 145477 exon 143323 142333

Allele GB:AC018755_2 225 142531 142531 G>T source isSNP SNP00064738 consequence HASl_cds.l 224 3'

Allele GB:AC018755_2 225 147775 147775 G>T source dbSNP gnl | dbSNP | ss715930_allele consequence HASl_cds.l 224 Intron

Allele GB:AC018755_2 225 149089 149089 A>G source isSNP SNP00096015 consequence HASl_cds.l 224 Missense 14-14 OR

Allele GB:AC018755_2 225 149293 149293 OG source dbSNP gnl | dbSNp| ss713606_allele consequence HASl_cds.l 224 Intron GIF HASl-genomic-rev.gif TABLE 1 (Cont.)

HAS2

Full name : hyaluronan synthase 2

Link : HAS2_link_cdna

Subsequence GB:NM_005328 3003 #227 CDS GB:NM_005328.1 1659 bp #228

ORF 536 2194 Allele GB:NM_005328 227 381 381 A>G source isSNP SNP00072998 consequence GB:NM_005328.1 228 5'

Allele GB:NM_005328 227 1357 1357 OT source isSNP SNP00104961 consequence GB:NM_005328.1 228 Missense 274-274 F>L

GIF HAS2-cdna-fwd.gif

HSPG2

Full name : heparan sulfate proteoglycan 2

Link : HSPG2_link_cdna

Subsequence GB:NM_005529_2 1 13793 #229 CDS GB:NM_005529_2.1 13182 bp #230

ORF 41 13222 Allele GB:NM_005529_2 229 2155 2155 A>G source isSNP SNP00054627 consequence GB:NM_005529_2.1 230 Silent 705-705

Allele GB:NM_005529_2 229 2340 2340 A>G source isSNP SNP00054628 consequence GB:NM_005529_2.1 230 Missense 767-767 S>N

Allele GB:NM_005529_2 229 3603 3603 A>G source isSNP SNP00109135 consequence GB:NM_005529_2.1 230 Missense 1188-1188 R>Q

Allele GB:NM_005529_2 229 3734 3734 A>G source isSNP SNP00109136 consequence GB:NM_005529_2.1 230 Missense 1232-1232 G>S

Allele GB:NM_005529_2 229 3943 3943 A>G source isSNP SNP00054629 consequence GB:NM_005529_2.1 230 Silent 1301-1301 V

Allele GB:NM_005529_2 229 4032 4032 A>G source isSNP SNP00054630 consequence GB:NM_005529_2.1 230 Missense 1331-1331 G>D

Allele GB:NM_005529_2 229 4554 4554 A>G source isSNP SNP00109138 consequence GB:NM__005529_2.1 230 Missense 1505-1505 V>A

Allele GB:NM_005529_2 229 7042 7042 A>G source isSNP SNP00048871 consequence GB:NM_005529_2.1 230 Silent 2334-2334 N

Allele GB:NM_005529_2 229 7503 7503 A>G source isSNP SNP00109139 consequence GB:NM_005529_2.1 230 Missense 2488-2488 S>L

Allele GB:NM_005529_2 229 9548 9548 A>G source isSNP SNP00109140 consequence GB:NM__005529_2.1 230 Missense 3170-3170 T>A

Allele GB:NM_005529_2 229 10294 10294 A>G source isSNP SNP00109141 consequence GB:NM__005529_2.1 230 Silent 3418-3418 TABLE 1 (Cont.)

Allele GB:NM_005529. 2 229 10663 10663 A>G source isSNP SNP00109142 consequence GB:NM_ _005529_2.1 230 Silent 3541- -3541 V

Allele GB :NM_005529. _2 229 10941 10941 A>G source isSNP SNP00109143 consequence GB:NM_ _005529_2.1 230 Missense 3634- -3634 Q>R

Allele GB:NM_005529. _2 229 11233 11233 OT source isSNP SNP00009830 consequence GB:NM_ _005529_2.1 230 Silent 3731- -3731 V

Allele GB:NM_005529. _2 229 12358 12358 A>G source isSNP SNP00009831 consequence GB:NM_ _005529_2.1 230 Silent 4106- -4106

Allele GB:NM_005529. _2 229 12604 12604 A>G source isSNP SNP00038416 consequence GB:NM_ _005529_2.1 230 Silent 4188- -4188

GIF HSPG2-cdna-fwd.gif

IBSP

Full name : IBSP

Link : IBSP link_cdna

Subsequence GB:HUMSIALO 1 1037 #231

CDS GB:HUMSIAL0.1 954 bp #232

ORF 72 1025

Allele GB:HUMSIALO 231 494 494 A>G source isSNP SNP00065793 consequence GB:HUMSIALO.1 232 Silent 141- -141 N

Allele GB:HUMSIALO 231 655 655 A>G source isSNP SNP00065794 consequence GB:HUMSIALO.1 232 Missense 195- -195 G>E

Allele GB:HUMSIALO 231 709 709 A>G source isSNP SNP00018906 consequence GB:HUMSIALO.1 232 Missense 213- -213 OD

GIF IBSP-!cdna-fwd.gif

Link : IBSP. _link_genomic

Subsequence GB:HUMBNSP01 1 2415 #233

Subsequence GB:HUMBNSP02 2516 3359 #234

Subsequence GB:HUMBNSP03 3460 5094 #235

Subsequence GB:HUMBNSP04 5195 9497 #236

Subsequence IBSP_cds .1 2863 7195 #237

CDS IBSP_ιcds.l 954 bp 6 exons #237 exon 2863 2916 exon 3009 3059 exon 3158 3235 exon 3571 3633 exon 5882 6040 exon 6647 7195

Allele GB:HUMBNSP04 236 1631 1631 A>G source isSNP SNP00065794 consequence IBSP_cds.l 237 Missense 195- -195 E E>G

Allele GB:HUMBNSP04 236 1685 1685 A>G source isSNP SNP00018906 consequence IBSP_cds.l 237 Missense 213-213 G>D

GIF IBSP-genomic-fwd.gif TABLE 1 (Cont.)

IER3

Full name : immediate early response

Link : IER3_link_cdna

Subsequence GB:Y14551_1 1 1230 #238

CDS GB:Y14551_1.1 471 bp #239

ORF 12 482 Allele GB:Y14551_1 238 838 838 A>G source isSNP SNP00052893 consequence GB:Y14551_1.1 239 GIF IER3-cdna-fwd.gif Link : FL_758754_link_genomic

Subsequence GB:AC006165 1 44118 #240 Subsequence GB:AC006165_2619577CD1 14601 15183 #241 Subsequence FL_2619577_mrna_build.1 14585 15920 #242 mRNA FL_2619577_mrna_build.l 1224 bp 2 exons #242 exon 14585 14810 exon 14923 15920

CDS GB:AC006165_2619577CD1 471 bp 2 exons #241 exon 14601 14810 exon 14923 15183

Allele GB:AC006165 240 15539 15539 A>G source isSNP SNP00052893 consequence GB:AC006165_2619577CD1 241

GIF IER3-genomic-fwd.gif

IHH

Full name : IHH

Link : IHH_link_σdna

Subsequence GB :HUMIHH 1277 #243 CDS GB:HUMIHH.2 939 bp #244

ORF 2 940 Allele GB:HUMIHH 243 457 457 A>G source isSNP SNP00097225 consequence GB:HUMIHH.2 244 Silent 152-152 GIF IHH-cdna-fwd.gif Link : IHH_link_genomic Subsequence IHH_cds .1 1 1469 #245 Subsequence GB:AB010092_1 1 315 #246 Subsequence GB:AB018075_1 416 698 #247

Subsequence GB:AB018076 1 799 1481 #248 CDS IHH_cds 1236 bp exons #245 exon 1 315 exon 426 687 exon 811 1469 Allele GB:AB018075_1 247 194 194 A>G source wetSNP GB:AB018075. l.vl94.G>A consequence IHH_cds .1 245 Missense 167-167 A>T Allele GB:AB018076_1 248 188 188 A>G source isSNP SNP00097225 consequence IHH_cds .1 245 Silent 251-251 GIF IHH-genomic-fwd.gif TABLE 1 (Cont.)

INHBA

Full name : inhibin, beta A

Link : FL_3526170_link_cdna

Subsequence FN:3526170CBl 1 1620 #249 CDS FN.3526170CB1.1 1281 bp #250

ORF 216 1496 Allele FN:3526170CB1 249 607 607 G>T source isSNP SNP00068777 consequence FN: 3526170CB1.1 250 Missense 131-131 T>K GIF INHBA-cdna-fwd.gif Link : FL_3526170_link_genomic

Subsequence GB:AC005027 1 199878 #251 Subsequence GB:AC005027_3526170CD1 16865 54957 #252 Subsequence FL_3526170_mrna_build.1 14163 55081 #253 mRNA FL_3526170_mrna_build.l 1620 bp . 3 exons #253 exon 14163 14234 exon 16722 17252 exon 54065 55081 CDS GB:AC005027_3526170CD1 1281 bp 2 exons #252 exon 16865 17252 exon 54065 54957 Allele GB:AC005027 251 16377 16377 A>G source dbSNP gnl | dbSNP | ss577365_allele source dbSNP gnl j dbSNpj ss588511_allele consequence GB:AC005027_3526170CD1 252 ' 5'

GIF INHBA-genomic-fwd.gif

IRS1

Full name : Insulin receptor substrate 1

Link : IRSl_link_cdna

Subsequence EM:S62539 1 5828 #254 CDS EM:S62539.1 3729 bp #255

ORF 1021 4749 Allele EM:S62539 254 3388 3388 A>G source isSNP SNP00067005 consequence EM:S62539.1 255 Missense 790-790 R>C

Allele EM:S62539 254 3887 3887 A>G source isSNP SNP00114530 consequence EM:S62539.1 255 Missense 956-956 E>G

Allele EM:S62539 254 5156 5156 OT source isSNP SNP00067006 consequence EM:S62539.1 255

GIF IRSl-cdna-fwd.gif Link : IRSl_link_genomic

Subsequence EM:S85963 100 6251 #256

Subsequence IRSl_cds.l 680 4411 #257

Subsequence IRSl__mrna_build.1 100 4432 #25

CDS IRSl_cds.l 3732 bp 1 exon #257 exon 680 4411 mRNA IRSl_mrna_build.l 4333 bp P 1 exon #258 exon 100 4432 TABLE 1 (Cont.)

Allele EM:S85963 256 850 850 A>G source wetSNP EM:S85963.v850.OT consequence IRSl_cds .1 257 Silent 90-! .0 I

Allele EM:S85963 256 1285 1285 A>G source wetSNP EM:S85963.vl285. .G>A consequence IRSl_cds.l 257 Silent 235- -235

Allele EM:S85963 256 1783 1783 A>G source wetSNP EM:S85963.vl783. .T>C consequence IRSl_cds .1 257 Silent 401- -401 H

Allele EM:S85963 256 2023 2023 A>G source wetSNP EM:S85963.v2023. .OT consequence IRSl_cds.l 257 Silent 481- -481 N

Allele EM:S85963 256 2117 2117 OG source wetSNP EM:S85963.v2117. .G>C consequence IRSl_cds.l 257 Missense 513- -513 A>P

Allele EM:S85963 256 2697 2697 A>G source wetSNP EM:S85963.v2697 .G>A consequence IRSl_cds .1 257 Missense 706- -706 G>D

Allele EM:S85963 256 2941 2941 A>G source wetSNP ΞM:S85963.v2941 .T>C consequence IRSl_cds .1 257 Silent 787- -787 H

Allele EM:S85963 256 2951 2951 A>G source isSNP SNP00 067005 consequence IRSl_cds .1 257 M Miisssseennssee 7 79911-- -779911 R>C

Allele EM:S85963 256 2995 2995 A A>>GG source wetSNP EM:S85963.v2995.A>G consequence IRSl_cds .1 257 Silent 805-805 A

Allele EM:S85963 256 3035 3035 OG source wetSNP EM:S85963.v3035.G>C consequence IRSl_cds.l 257 Missense 819-819 G>R

Allele EM:S85963 256 3262 3262 OG source wetSNP EM:S85963.v3262.G>C consequence IRSl_cds.l 257 Silent 894-894

Allele EM:S85963 256 3349 3349 A>G source wetSNP EM:S85963.v3349.G>A consequence IRSl_cds .1 257 Silent 923-923 R

Allele EM:S85963 256 3450 3450 A>G source isSNP SNP00114530 consequence IRSl_cds .1 257 Missense 957-957 E>G

Allele ΞM:S85963 256 3494 3494 A>G source wetSNP EM:S85963.v3494.G>A consequence IRSl_cds.l 257 Missense 972-972 OR

Allele EM:S85963 256 4053 4053 A>G source wetSNP EM: S85963.V4053.OA consequence IRSl cds.l 257 Missense 1158-115E OE

GIF IRSl-genomic-fwd.gif

JUN

Full name : v-jun avian sarcoma virus 17 oncogene homolog

Link : JUN_link_genomic

Subsequence UN_σds .1 9468 8473 #259 Subsequence GB: L136985_1 1 151212 #260

Subsequence JUN mrna build.l 58 8473 #261 TABLE 1 (Cont.)

CDS JUN_cds.l 996 bp 1 exon #259 exon 9468 8473 mRNA σUN_mrna_build.l 996 bp 1 exon #261 exon 9468 8473 GIF JUN-genomic-rev.gif

KJ_OAll

Full name : KIAA1253

Link : FL_2135776_link_cdna

Subsequence FN:2135776CB1 3129 #262 CDS FN.2135776CB1.1 1197 bp #263

ORF 256 1452 Allele FN:2135776CB1 262 59 59 OG source isSNP SNP00100733 consequence FN:2135776CB1.1 263 5'

Allele FN:2135776CB1 262 1352 1352 A>G source isSNP SNP00116557 consequence FN:2135776CB1.1 263 Missense 366-366 Q>R

Allele FN:2135776CB1 262 1477 1477 A>G source isSNP SNP00042286 consequence FN:2135776CB1.1 263 3'

Allele FN:2135776CB1 262 1489 1489 A>G source isSNP SNP00042287- consequence FN:2135776CB1.1 263 3'

Allele FN:2135776CB1 ^' 262 1667 1667 A>G source isSNP SNP00011480 consequence FN: 2135776CB1.1 263 3'

Allele FN:2135776CB1 262 1710 1710 A>G source isSNP SNP00011481 consequence FN: 2135776CB1.1 263 3'

Allele FN:2135776CB1 262 1838 1838 A>G source isSNP SNP00011482 consequence FN: 2135776CB1.1 263 3'

Allele FN:2135776CB1 262 2589 2589 A>G source isSNP SNP00003671 consequence FN: 2135776CB1.1 263 GIF KJ_OAll-cdna-fwd.gif Link : FL_2135776_link_genomic

Subsequence GB:HS425C14 1 160203 #264 Subsequence GB:HS425C14_2135776CD1 55766 42255 #265 Subsequence FL_2135776_mrna_build.1 69012 40562 #266 Subsequence KJ_OAll_cds .1 55766 51052 #267 CDS GB:HS425C14_2135776CD1 1197 bp 9 exons #265 exon 55766 55731 exon 53861 53692 exon 51441 51362 exon 51118 50981 exon 49268 49099 exon 48965 48875 exon 44476 44332 exon 44215 43985 exon 42390 42255 mRNA FL_2135776_mrna_build.l 3119 bp 10 exons #266 TABLE 1 (Cont.) exon 69012 68910 exon 55892 55731 exon 53861 53692 exon 51441 51362 exon 51118 50981 exon 49268 49099 exon 48965 48875 exon 44476 44332 exon 44215 43985 exon 42390 40562

CDS KJ_OAll_cds.1 273 bp 3 exons #267 exon 55766 55731 exon 53861 53692 exon 51118 51052

Allele GB:HS425C14 264 41092 41092 A>G source isSNP SNP00003671 consequence GB:HS425C14.2135776CD1 265 consequence KJ_OAll_cds 1 267 3'

Allele GB:HS425C14 264 41843 41843 A>G source isSNP SNP00011482 consequence GB:HS425C14.2135776CD1 265 consequence KJ_OAll_cds 1 267 3'

Allele GB:HS425C14 264 41971 41971 A>G source isSNP SNP00011481 consequence GB:HS425C14.2135776CD1 265 consequence KJ_OAll_cds 1 267 3'

Allele GB:HS425C14 264 42014 42014 A>G source isSNP SNPO0011480 consequence GB:HS425C14_.2135776CD1 265 consequence KJ_OAll_cds 1 267 3'

Allele GB:HS425C14 264 42192 42192 A>G source isSNP ΞNP00042287 consequence GB:HS425C14.2135776CD1 265 consequence KJ_OAll_cds 1 267 3'

Allele GB:HS425C14 264 42204 42204 A>G source isSNP SNP00042286 source wetSNP GB:HS425C14, v42204.G>A consequence GB:HS425C14.2135776CD1 265 3' consequence KJ_OAll_cds 1 267 3'

Allele GB:HS425C14 264 42294 42294 OG source wetSNP GB:HS425C14. v42294.G>C source wetSNP GB:HS425C14, v42294.G>C consequence GB:HS425C14:_._2135776CD1 265 Silent 386-386 consequence KJ_OAll_σds.l 267

Allele GB:HS425C14 264 42329 42329 A>G source isSNP SNP00116557 consequence GB:HS425C14_2135776CD1 265 Missense 375-375

S>G consequence KJ_OAll_cds.l 267 3'

Allele GB:HS425C14 264 44297 44297 A>G source wetSNP GB:HS425C14.v44297.T>C consequence GB:HS425C14_2135776CD1 265 Intron consequence KJ_0A1l_cds .1 267 3 '

Allele GB:HS425C14 264 55697 55697 A>G TABLE 1 (Cont.) source wetSNP GB:HS425Cl4.v55697.C>T consequence GB:HS425C14_2135776CD1 265 Intron consequence KJ_OAll_cds.l 267 Intron

Allele GB :HS425C14 264 68954 68954 OG source isSNP SNP00100733 consequence GB:HS425C14_2135776CD1 265 consequence KJ OA11 cds.l 267 5'

GIF KJ_OAll-genomic-rev.gif

KJ_OA2

Link : KJ_OA2_link_cdna

Subsequence LG: 244552.16 1 1825 #268 Allele LG:244552.16 268 1476 1476 OT source isSNP SNP00098862

KJ_OA21

Full name : FL project 2027624

Link : FL_2027624_link_cdna

Subsequence FN:2027624CB1 1 2173 #269 CDS FN.2027624CB1.1 1734 bp #270

ORF 4 1737 Allele FN:2027624CB1 269 881 881 OG source isSNP SNP00106459 consequence FN: 2027624CB1.1 270 Missense 293-293 T>R

Allele FN:2027624CB1 269 971 971 A>G source isSNP SNP00075286 consequence FN: 2027624CB1.1 270 Missense 323-323 T>I

Allele FN:2027624CB1 269 1092 1092 OG source isSNP SNP00106460 consequence FN:2027624CB1.1 270 Silent 363-363

Allele FN:2027624CB1 269 1254 1254 A>G source isSNP SNP00075287 consequence FN: 2027624CB1.1 270 Silent 417-417

Allele FN:2027624CB1 269 1374 1374 A>G source isSNP SNP00009699 consequence FN: 2027624CB1.1 270 Silent 457-457 T

Allele FN:2027624CB1 269 1392 1392 A>G source isSNP SNP00097916 consequence FN: 2027624CB1.1 270 Silent 463-463

Allele FN:2027624CB1 269 1623 1623 A>G source isSNP SNP00009700 consequence FN: 2027624CB1.1 270 Silent 540-540 GIF KJ_OA21-cdna-fwd.gif Link : FL_1250708_link_genomic

Subsequence GB:HS453C12 1 147620 #271 Subsequence GB:HS453C12_1394592CD1 87967 109084 #272 Subsequence GB:HS453C12_2027624CD1 20194 10528 #273 Subsequence FL_1394592_mrna_build.l 87945 110578 #274 Subsequence FL_2027624_mrna_build.l 20197 6152 #275 Subsequence OA21 cds.l 20194 17050 #276 mRNA FL_2027624 mrna build.1 Ψ² bp 13 exons #275 TABLE 1 (Cont.) exon 20197 20008 exon 19834 19657 exon 17499 17372 exon 17056 16956 exon 16847 16761 exon 16215 16128 exon 16019 15922 exon 15823 15658 exon 14968 14768 exon 12135 11970 exon 11855 11772 exon 10777 10110 exon 6168 6152

OA21_ .eds .1 372 bp 3 exons #276 exon 20194 20008 exon 19834 19657 exon 17056 17050

CDS GB:HS453C12_2027624CD1 1734 bp 12 exons #273 exon 20194 20008 exon 19834 19657 exon 17499 17372 exon 17056 16956 exon 16847 16761 exon 16215 16128 exon 16019 15922 exon 15823 15658 exon 14968 14768 exon 12135 11970 exon 11855 11772 exon 10777 10528

Allele GB:HS453C12 271 10642 10642 A>G source « isSNP SNP00009700 source wetSNP GB:HS453C12.vl0642.A>G source wetSNP GB:HS453C12.vl0642.A>G consequence OA21_cds.l 276 3' consequence GB:HS453C12_2027624CD1 273 Silent 540-540

Y Allele GB:HS453C12 271 11206 11206 A>G source dbSNP gnl|dbSNP|ss979258_allele consequence OA21_cds.l 276 3' consequence GB:HS453C12_2027624CD1 273 Intron

Allele GB:HS453C12 271 11999 11999 A>G source isSNP SNP00009699 source wetSNP GB:HS453C12.vll999.C>T source wetSNP GB:HS453C12.vll999.C>T consequence OA21_σds.l 276 3' consequence GB:HS453C12_2027624CD1 273 Silent 457-457

T Allele GB:HS453C12 271 13494 13494 A>G source isSNP SNP00095042 consequence OA21_cds.l 276 3' consequence GB:HS453C12_2027624CD1 273 Intron

Allele GB:HS453C12 271 14913 14913 OG source isSNP SNP00106460 consequence OA21_cds.l 276 3' TABLE 1 (Cont.) consequence GB:HS453C12_2027624CDl 273 Silent 363-363

L Allele GB:HS453C12 271 15723 15723 A>G source isSNP SNP00075286 consequence OA21_σds.l 276 3' consequence GB :HS453C12_2027624CD1 273 Missense 323-323

T>l

GIF KJ_OA21-genomic-rev.gif

KJ__OA29

Link : KJ_OA29 link_cdna

Subsequlence LG: 199489.1 1 3318 #277

Allele LG:199489 .1 277 544 544 A>G source isSNP SNP00005297

Allele LG:199489 .1 277 695 695 A>G source isSNP SNP00121995

Allele LG:199489 .1 277 971 971 A>G source isSNP SNP00047679

Allele LG:199489 .1 277 1312 1312 A>G source isSNP SNP00005298

Allele LG:199489 .1 277 1445 1445 A>G source isSNP SNP00027647

Allele LG:199489 .1 277 2370 2370 A>G source isSNP SNP00005297

Allele LG:199489 .1 277 2521 2521 A>G source isSNP SNP00121995

Allele LG:199489. .1 277 2797 2797 A>G source isSNP SNP00047679

Allele LG:199489. .1 277 3138 3138 A>G source isSNP SNP00005298

Allele LG:199489 .1 277 3271 3271 A>G source isSNP SNP00027647

KJ_OA3

Link : KJ OA3 link cdna

Subsequenc :e LG:153511.1 1 1628 #278

Allele LG:153511.1 278 395 395 A>G source isSNP SNP00003503

Allele LG:153511.1 278 1101 1101 A>G source isSNP SNP00113687

KJ_OA31

Link : KJ_OA31_link_cdna

Subsequenc :e LG:200972.2 1 2192 #279

Allele LG:200972.2 279 366 366 OG source isSNP SNP00099556

Allele LG:200972.2 279 836 836 A>G source isSNP SNP00015954

Allele LG:200972.2 279 1037 1037 A>G TABLE 1 (Cont.) source isSNP SNP00015955

Allele LG:200972, .2 279 1361 1361 A>G source isSNP SNP00000598

Allele LG:200972 .2 279 1697 1697 A>G source isSNP SNP00000599

Allele LG:200972 .2 279 1975 1975 A>G source isSNP SNP00067907

Allele LG:200972 .2 279 2027 2027 A>G source isSNP SNP00067908

KJ_OA33

Full name : cardiotrophin-like cytokine

Link : FL_1676240_link_genomic

Subsequence GB:AC005849_1 1 169144 #280 Subsequence KJ_OA33_cds .1 151862 143455 #281 Subsequence KJ_OA33_mrna_build.1 151907 142489 #282

KJ_OA33_cds.l 678 bp 3 exons #281 exon 151862 151847 exon 145945 145779 exon 143949 143455 mRNA KJ_OA33. _mrna_build.1 1689 bp 3 exons #282 exon 151907 151847 exon 145945 145779 exon 143949 142489

GIF KJ_OA33-genomic-rev.gif

KJ_OA39

Link : KJ_OA39_link_cdna

Subsequence LG: 293953.1 1 940 #283 Allele LG:293953.1 283 679 679 OT source isSNP SNP00110603

KJ_OA6

Full name : FL project 2840746

Link : FL_818498_link_genomic

Subsequence GB:AC005598 1 190000 #284

Subsequence GB:AC005598_2840746CDl 132700 133368 #285

Subsequence FL_2840746_mrna_b ild.1 132672 135584 #286

CDS GB:AC005598_2840746CD1 669 bp 1 exon #285 exon 132700 133368 mRNA FL_2840746_mrna_build.l 1087 bp 2 exons #286 exon 132672 133391 exon 135218 135584

Allele GB:AC005598 284 132689 132689 A>G source isSNP SNP00005520 consequence GB:AC005598_2840746CDl' 285 5'

Allele GB:AC005598 284 132843 132843 A>G source wetSNP GB:AC005598.vl32843.OT consequence GB:AC005598_2840746CD1 285 Silent 48-48 S TABLE 1 (Cont.)

Allele GB:AC005598 284 132878 132878 A>G source wetSNP GB AC005598.vl32878.G>A consequence GB:AC005598_2840746CD1 285 Missense 60-60 R>H

Allele GB:AC005598 284 132951 132951 A>G source wetSNP GB AC005598.vl32951.C>T consequence GB:AC005598_2840746CD1 285 Silent 84-84 F

Allele GB:AC005598 284 132967 132967 A>G source wetSNP GB AC005598.vl32967.C>T consequence GB:AC005598_2840746CD1 285 Missense 90-90 P>S

Allele GB:AC005598 284 133103 133103 G>T source wetSNP GB AC005598.vl33103.G>T consequence GB:AC005598 2840746CD1 285 Missense 135-135

G>V

Allele GB:AC005598 284 133481 133481 A>G source wetSNP GB:AC005598.vl33481.OT consequence GB:AC005598_2840746CD1 285 3' GIF KJ_OA6-genomiσ-fwd .gif

KJ_oagba3

Link : KJ_oagba3_link_cdna

Subsequence LG:215642.2 1 2849 #287

Allele LG:215642.2 287 1475 1475 A>G source isSNP SNP00041601

Allele LG:215642.2 287 1963 1963 A>G source isSNP SNP00010951

LIF

Full name : leukemia inhibitory factor

Link : LIF_link_cdna

Subsequeance GB:LIF 1 3848 #28!

CDS GB: LIF.l 609 bp #289

ORF 45 653

Allele GB:LIF 288 1183 1183 G>T source isSNP SNP00036337 consequence GB:LIF.l 289 3'

Allele GB:LIF 288 1572 1572 A>G source isSNP SNP00099092 consequence GB: LIF.l 289 3'

Allele GB:LIF 288 1996 1996 OG source isSNP SNP00099093 consequence GB: LIF.l 289 3'

Allele GB :LIF 288 2062 2062 OT source isSNP SNP00099094 consequence GB:LIF.l 289 3'

Allele GB:LIF 288 2404 2404 A>G source isSNP SNP00099095 consequence GB:LIF.l 289 3'

Allele GB :LIF 288 3156 3156 A>G source isSNP SNP00036338 consequence GB: LIF.l 289 3'

Allele GB :LIF 288 3582 3582 A>G TABLE 1 (Cont.) source isSNP SNP00008778 consequence GB.LIF.l 289 GIF LIF-cdna-fwd.gif Link : OSM_link_genomic

Subsequence GB:AC004264 1 47188 #290

Subsequence LIF_cds.l 11398 8354 #291

Subsequentce LIF_mrna_build.1 11442 5156 #292

CDS LIF_cds.l 609 bp 3 exons #291 exon 11398 11380 exon 9636 9458 exon 8764 8354 mRNA LIF_mrna_build.l 3851 bp 3 ex< #292 exon 11442 11380 exon 9636 9458 exon 8764 5156

Allele GB:AC004264 290 5420 5420 A>G source isSNP SNP00008778 consequence LIF_cds.l 291 3'

Allele GB:AC004264 290 5846 5846 A>G source isSNP SNP00036338 consequence LIF_cds.l 291 3'

Allele GB:AC004264 290 6598 6598 A>G source isSNP SNP00099095 consequence LIF_cds.l 291 3'

Allele GB:AC004264 290 6940 6940 OT source isSNP SNP00099094 consequence LIF_cds.l 291 3'

Allele GB:AC004264 290 7006 7006 OG source isSNP SNP00099093 consequence LIF_cds.l 291 3'

Allele GB:AC004264 290 7435 7435 A>G source isSNP SNP00099092 consequence LIF_cds.l 291 3'

Allele GB:AC004264 290 7824 7824 G>T source isSNP SNP00036337 consequence LIF_cds.l 291 3'

GIF LIF-genomic-rev.gif

LUM

Full name : lumican Link : FL_2676170_link_genomic Subsequence GB :AC007115_1 1 180821 #293 Subsequence GB:AC007115_1.3128106CD1 87417 92234 #294 Subsequence FL_3128106_mrna_b ild.1 84719 92839 #295 mRNA FL_3128106_mrna_build.l 1926 bp 3 exons #295 exon 84719 84998 exon 87396 88278 exon 92077 92839 CDS GB:AC007115_1_3128106CD1 1020 bp exons #294 exon 87417 88278 exon 92077 92234 Allele GB:AC007115 1 293 89050 89050 A>G source dbSNP gnl|dbSN_tE|ss852530_allele TABLE 1 (Cont.) source dbSNP gnl |dbSNP| ss897123_allele consequence GB:AC007115_1_3128106CD1 294 Intron

Allele GB :AC007115_1 293 89249 89249 A>G source dbSNP gnl | dbSNP | ss855039_allele consequence GB:AC007115_l_3128106CDl 294 Intron

GIF LUM-genomic-fwd.gif

METTL1

Full name : methyltransferase-like

Link : METTL1 link cdna

Subsequence GB:Y18643_1 1 1292 #296

CDS GB:Y18643_1.1 831 bp #297

ORF 49 879

Allele GB:Y18643_1 296 345 345 A>G source isSNP SNP00098761 consequence GB:Y18643_1.1 297 Silent 99-99 P

Allele GB:Y18643_1 296 919 919 A>G source isSNP SNP00003825 consequence GB:Y18643_1.1 297

GIF METTLl-cdna-fwd.gif

MMPl

Full name : matrix metalloproteinase 1

Link : MMPl link cdna

Subsequence EM:HSC0LL1 1 1970 #298

Allele EM:HSC0LL1 298 383 383 A>G source isSNP SNP00009627

Allele EM:HSC0LL1 298 714 714 A>G source isSNP SNP00037857

Allele EM:HSCOLLl 298 745 745 A>G source isSNP SNP00037858

Allele- EM:HSCOLL1 298 1522 1522 A>G source isSNP SNP00009628

Allele EM:HSCOLL1 298 1541 1541 A>G source isSNP SNP00009629

Allele EM:HSC0LL1 298 1662 1662 A>G source isSNP SNP00009630

Allele EM:HSC0LL1 298 1747 1747 A>G source isSNP SNP00009631 nk : MMP1_ _link_genomic

Subsequence GB:HSU78045 1 81826 #299

Subsequence MMPl_cds .1 11905 4225 #300

Subsequence MMPl_mrna_build.l 11973 3733 #301

CDS MMPl_ eds .1 1410 bp 10 exons #300 exon 11905 11801 exon 11314 11070 exon 10976 10828 exon 10603 10478 exon 9421 9266 exon 9105 8988 exon 6551 6418 TABLE 1 (Cont.) exon 5308 5146 exon 4619 4516 exon 4334 4225 mRNA MMPl_mrna_bulild.l 1970 bp 10 exons #301 exon 11973 11801 exon 11314 11070 exon 10976 10828 exon 10603 10478 exon 9421 9266 exon 9105 8988 exon 6551 6418 exon 5308 5146 exon 4619 4516 exon 4334 3733

Allele GB:HSU78045 299 3956 3956 A>G source isSNP SNP00009631 consequence MMPl_cds .1 300 3'

Allele GB:HSU78045 299 4041 4041 A>G source isSNP SNP00009630 consequence MMPl_cds .1 300 3'

Allele GB:HSU78045 299 4162 4162 A>G source isSNP SNP00009629 consequence MMPl_cds .1 300 3'

Allele GB:HSU78045 299 4181 4181 A>G source isSNP SNP00009628 consequence MMPl_cds .1 300 3'

Allele GB:HSU78045 299 4517 4517 A>G source wetSNP GB:HSU78045.v4517 .A>G consequence MMPl_cds .1 300 Silent 433-433 D

Allele GB:HSU78045 299 4661 4664 CATOCG source wetSNP GB :HSU78045. 4661. . CATOCG consequence MMPl_cds .1 300 Intron

Allele GB:HSU78045 299 4677 4677 A>G source wetSNP GB:HSU78045.v4677 .G>A consequence MMPl_cds .1 300 Intron

Allele GB:HSU78045 299 5198 5198 A>G source wetSNP GB:HSU78045.v5198 .A>G consequence MMPl_σds .1 300 Missense 382-382 S>P

Allele GB:HSU78045 299 6586 6586 A>G source wetSNP GB:HSU78045.v6586 .T>C consequence MMPl_cds .1 300 Intron

Allele GB:HSU78045 299 9056 9056 A>G source wetSNP GB:HSU78045.v9056. .OT consequence MMPl_cds .1 300 Silent 277-277

Allele GB:HSU78045 299 9120 9120 A>G source wetSNP GB:HSU78045.v9120 .A>G consequence MMPl_cds .1 300 Intron

Allele GB:HSU78045 299 9126 9126 A>G source wetSNP GB:HSU78045.v9126. .OA consecjuence MMPl_cds .1 300 Intron

Allele GB:HSU78045 299 9205 9205 A>G source wetSNP GB:HSU78045.v9205 .T>C consecjuence MMPl_cds .1 300 Intron

Allele GB-.HSU78045 299 9247 9247 A>G source wetSNP GB:HSU78045.v9247 .T>C TABLE 1 (Cont.) consequence MMPl_cds .1 300 Intron

Allele GB:HSU78045 299 9365 9365 OT source wetSNP GB:HSU78045.v9365 .OT consequence MMPl_cds .1 3 30000 MMiisssseennssee 2 22288--222288 H>N

Allele GB:HSU78045 299 9370 9370 A>G source isSNP SNPOC 37858 consequence MMPl_cds .1 3 30000 MMiisssseennssee 2 22266--222266 L>P

Allele GB:HSU78045 299 11105 11105 A>G source isSNP SNP00009627 source wetSNP GB:HSU78045.vlll05.OT consequence MMPl_cds.l 300 Silent 105-105 G

GIF MMPl-genomic-rev.gif

MMPl3

Full name : MMP13

Link : MMP13_link_genomic

Subsequence MMP13_cds.l 141623 159614 #302 Subsequence GB:AP000789_1 1 201766 #303 CDS MMP13_cds.l 957 bp 7 exons #302 exon 141629 141779 exon 141956 142081 exon 144063 144224 exon 146009 146126 exon 147078 147211 exon 157208 157367 exon 159509 159614 Allele GB:AP000789__1 303 141614 141614 OG source wetSNP GB:AP000789_l.vl41614.OG consequence MMP13_cds. 302 5'

Allele GB:AP000789_1 303 141875 141875 OT source wetSNP GB : AP000789_l . vl41875 . A consequence MMP13_cds . 302 Intron

Allele GB:AP000789_1 303 147095 147095 A>G source wetSNP GB : AP000789_1 . V147095 . A>G consequence MMP13_cds. 302 Missense 192-192 H>R

Allele GB:AP000789_1 303 157231 157231 OG source wetSNP GB : AP000789_l . vl57231 . OC consequence MMP13__cds. 302 Missense 239-239 OR Allele GB:AP000789_1 303 157325 157325 A>G source wetSNP GB : AP000789_l .vl57325 . A>G consequence MMP13_cds. 302 Missense 270-270 D>G Allele GB:AP000789_1 303 159631 159631 A>G source wetSNP GB : AP000789_l . vl59631 . OT consequence MMPl3_cds. 302 3 ' Allele GB:AP000789_1 303 159644 159644 OG source wetSNP GB : AP000789_l . vl59644 . G>C consequence MMPl3_cds . 302 3 ' GIF MMP13-genomic-fwd.gif

MMP14 Full name MMP14 TABLE 1 (Cont.)

Link : MMP14_link_cdna

Subsequence GB:HUMMTMMP 1 3403 #304

CDS GB:HUMMTMMP.1 1749 bp #305

ORF 112 1860 Allele GB :HUMMTMMP 304 133 133 A>G source isSNP SNP00107954 consequence GB :HUMMTMMP.1 305 Missense 8-8 S S>P

Allele GB :HUMMTMMP 304 580 580 A>G source isSNP SNP00107955 consequence GB:HUMMTMMP.1 305 Silent 157- -157

Allele GB:HUMMTMMP 304 888 888 OG source isSNP SNP00093383 consequence GB:HUMMTMMP.1 305 Silent 259- -259

Allele GB :HUMMTMMP 304 966 966 A>G source isSNP SNP00055171 consequence GB :HUMMTMMP.1 305 Silent 285- -285 G

Allele GB :HUMMTMMP 304 1243 1243 A>G source isSNP SNP00107956 consequence GB :HUMMTMMP.1 305 Missense 378- -378 K>E

Allele GB :HUMMTMMP 304 1264 1264 OG source isSNP SNP00107957 consequence GB :HUMMTMMP.1 305 Missense 385- -385 D>H

Allele GB :HUMMTMMP 304 1944 1944 A>G source isSNP SNP00060446 consequence GB :HUMMTMMP.1 305 3' GIF MMP14-cdna-fwd.gif Link : MMP14_link_genomic

Subsequence MMP14_cds.l 132034 141254 #306 Subsequence GB:AL133448_3 1 173805 #307 Subsequence MMP14_mrna_build.1 131922 142801 #308 CDS MMP14_cds.l 1749 bp 10 exons #306 exon 132034 132141 exon 136706 136854 exon 137128 137250 exon 137625 137932 exon 138472 138633 exon 138925 139085 exon 139586 139724 exon 139845 139995 exon 140466 140581 exon 140923 141254 mRNA MMPl4_mrna_build.1 3408 bp 10 exo: #308 exon 131922 132141 exon 136706 136854 exon 137128 137250 exon 137625 137932 exon 138472 138633 exon 138925 139085 exon 139586 139724 exon 139845 139995 exon 140466 140581 exon 140923 142801

Allele GB:AL133448_. _3 307 132055 132055 A>G source isSNP SNP00107954 consequence MMP14_cds .1 306 Missense 8-8 P>S TABLE 1 (Cont.)

Allele GB:AL133448_ .3 307 137049 137051 TTA>TA source wetSNP GB :AL133448_3.vl37049.TTA>TA consequence MMP14_cds .1 306 Intron

Allele GB:AL133448_.3 307 137713 137713 A>G source isSNP SNP00 107955 consequence MMP14_cds .1 306 Silent 157 -157 1

Allele GB:AL133448_.3 307 138406 138406 A>G source wetSNP GB :AL133448_3.vl38406.OA consequence MMP14_cds .1 306 Intron

Allele GB:AL133448_ 3 307 138560 138560 OG source isSNP SNP00 093383 source wetSNP GB:AL133448_3.vl38560. OG consequence MMP14_cds .1 306 Silent 259 -259 P

Allele GB:AL133448_ 3 307 138653 138653 A>G source wetSNP GB:AL133448_3.vl38653.OA consequence MMP14_σds .1 306 Intron

Allele GB:AL133448_.3 307 139639 139639 A>G source wetSNP GB:AL133448_3.vl39639. G>A consequence MMPl4_cds .1 306 Missense 355 -355 M>I

Allele GB:AL133448_.3 307 139981 139981 A>G source wetSNP GB:AL133448_3.vl39981. OT consequence MMP14_σds .1 306 Silent 429 -429 F

Allele GB:AL133448__.3 307 139986 139986 A>G source wetSNP GB:AL133448_3.vl39986. G>A consequence MMP14_cds .1 306 Missense 431 -431 R>H

Allele GB:AL133448_.3 307 141337 141337 A>G source isSNP SNP00 060446 consequence MMP14_cds.l 306 3'

GIF MMPl4 -genomic-fwd. gif

MMP2

Link : MMP2_link_cdna

Subsequence GB :HSMMPM2 3530 #309

CDS GB: HSMMPM2.1 2010 bp #310

ORF 49 2058

Allele GB:HSMMPM2 309 681 681 A>G source isSNP SNP00100004 consequence GB:HSMMPM2.1 310 Silent 211-211

Allele GB:HSMMPM2 309 1835 1835 A>G source isSNP SNP00100005 consequence GB :HSMMPM2.1 310 Missense 596-596 D>G

Allele GB:HSMMPM2 309 1851 1851 OT source isSNP SNP00075435 consequence GB:HSMMPM2.1 310 Missense 601-601 F>L

Allele GB :HSMMPM2 309 2717 2717 A>G source isSNP SNP00024650 consequence GB:HSMMPM2.1 310 3'

Allele GB:HSMMPM2 309 2922 2922 OG source isSNP SNP00024651 consequence GB:HSMMPM2.1 310

GIF MMP2-cdna-fwd.gif Link : MMP2_link_genomic

Subsequence MMP2_cds .1 175558 156463 TABLE 1 (Cont.)

Subsequence GB:AC012182_3 1 190117 #312 Subsequence MMP2_mrna_build.l 175606 155007 #313 CDS MMP2 cds.l 2010 bp 10 exons #311 exon 175558 175397 exon 164437 164289 exon 163643 163515 exon 162034 161727 exon 161372 161211 exon 160292 160039 exon 159678 159540 exon 158699 158549 exon 158397 158282 exon 156902 156463 mRNA MMP2_mrna_build.l 3514 bp 10 exons #313 exon 175606 175397 exon 164437 164289 exon 163643 163515 exon 162034 161727 exon 161372 161211 exon 160292 160039 exon 159678 159540 exon 158699 158549 exon 158397 158282 exon 156902 155007

Allele GB:AC012182_3 312 155598 155598 OG source isSNP SNP00024651 consequence MMP2_cds .1 311 3'

Allele GB:AC012182_3 312 155804 155804 A>G source isSNP SNP00024650 consequence MMP2_cds .1 311 3'

Allele GB:AC012182_3 312 156670 156670 OT source isSNP SNP00075435 consequence MMP2_cds .1 311 Missense 601-601 F>L

Allele GB:AC012182_3 312 156686 156686 A>G source isSNP SNP00100005 consequence MMP2_cds .1 311 Missense 596-596 D>G

Allele GB:AC012182_3 312 161842 161842 A>G source isSNP SNP00100004 consequence MMP2_cds .1 311 Silent 211-211

Allele GB:AC012182_3 312 163660 163660 A>G source wetSNP GB:AC012182_3.vl63660.G>A consequence MMP2_σds .1 311 Intron GIF MMP2-genomic-rev.gif

MMP3

Full name : matrix metalloproteinase 3

Link : MMP3 link_cdna

Subsequenc ;e EM:HSSTROMR 1 1801 #314

Allele EM:HSSTROMR 314 331 331 A>G source isSNP SNP00011525

Allele EM:HSSTROMR 314 382 382 A>G source isSNP SNP00113489

Allele EM:HSSTROMR 314 713 713 A>G TABLE 1 (Cont.) source : isSNP SNP00015044

Allele EM:HSSTROMR 314 976 976 A>G sourcei isSNP SNP00054705

Allele EM:HSSTROMR 314 1129 1129 A>G source isSNP SNP00011527

Link : MMP3_ .1ink_genomic

Subsequenc :e EM:HSU78045 100 81925 #315

Subsequenc :e MMP3_link_cds.l 57437 50020 #316

Subsequenc :e MMP3_mrna_build.l 57480 49696 #317

CDS MMP3_; Link_cd .s.l 1434 bp 10 exons #316 exon 57437 57333 exon 56806 56562 exon 56469 56321 exon 56182 56057 exon 54487 54323 exon 54146 54002 exon 53137 53004 exon 52604 52445 exon 51295 51192 exon 50120 50020 mRNA MMP3_mrna_b ild.1 1801 bp 10 exons #317 exon 57480 57333 exon 56806 56562 exon 56469 56321 exon 56182 56057 exon 54487 54323 exon 54146 54002 exon 53137 53004 exon 52604 52445 exon 51295 51192 exon 50120 49696

Allele EM:HSU78045 315 52375 52375 A>G source wetSNP EM:HSU78045.v52375.T>C consequence MMP3_link_cds.l 316 Silent 400-400

Allele EM:HSU78045 315 52411 52411 A>G source wetSNP EM:HSU78045.v52411.G>A consequence MMP3_link_cds.l 316 Silent 388-388

Allele EM:HSU78045 315 52489 52489 A>G source wetSNP EM:HSU78045.v52489.G>A consequence MMP3_link_cds.l 316 Silent 362-362

Allele EM:HSU78045 315 52527 52530 GAGT>GT source wetSNP EM:HSU78045.v52527.GAGT>GT consequence MMP3_link_cds.l 316 Intron

Allele EM:HSU78045 315 52586 52586 A>T source wetSNP EM:HSU78045.v52586.T>A consequence MMP3_link_cds.l 316 Intron

Allele EM:HSU78045 315 53771 53771 A>T source wetSNP EM:HSU78045.v53771.T>A consequence MMP3_link_cds.l 316 Intron

Allele EM:HSU78045 315 54077 54077 OG source wetSNP EM:HSU78045.v54077.OG consequence MMP3_link_cds.l 316 Intron

Allele EM:HSU78045 315 54187 54187 A>G source wetSNP EM:HSU78045.v54187.OT consequence MMP3_link ^k_-c^cd^ds^sι,kl, 316 Intron TABLE 1 (Cont.)

Allele ΞM:HSU78045 315 54402 54402 A>G source wetSNP EM:HSU78045 V54402.OT consequence MMP3_link_cds .1 316 Intron

Allele EM:HSU78045 315 56119 56119 A>G source wetSNP EM:HSU78045 V56119.0T consequence MMP3_link_cds .1 316 Intron

Allele EM:HSU78045 315 56507 56507 OG source wetSNP EM:HSU78045. V56507. C consequence MMP3_link_cds .1 316 Silent 102-102

Allele EM:HSU78045 315 56525 56525 A>G source isSNP SNP00011525 source wetSNP EM:HSU78045. v56525.G>A consequence MMP3_link_cds.1 316 Silent 96-96 D

Allele EM:HSU78045 315 56680 56680 A>G source wetSNP EM:HSU78045, V56680. T consequence MMP3_link_cds .1 316 Missense 45-45 E>K

GIF MMP3-genomic-rev.gif

MMP9

Full name : matrix metalloproteinase

Link : MMP9_link_cdna

Subsequence FN:522678CB1 2348 #318 CDS FN:522678CB1.1 2124 bp #319

ORF 33 2156 Allele FN:522678CB1 318 308 308 A>G source isSNP SNP00101082 consequence FN: 522678CB1.1 319 Silent 92-92 K

Allele FN:522678CB1 318 413 413 A>G source isSNP SNP00101083 consequence FN: 522678CB1.1 319 Silent 127-127 N

Allele FN:522678CB1 318 534 534 A>G source isSNP SNP00101084 consequence FN: 522678CB1.1 319 Missense 168-168 I>V

Allele FN:522678CB1 318 591 591 A>G source isSNP SNP00101085 consequence FN: 522678CB1.1 319 Missense 187-187 L>F

Allele FN:522678CB1 318 719 719 A>G source isSNP SNP00101086 consequence FN: 522678CB1.1 319 Silent 229-229

Allele FN:522678CB1 318 748 748 A>G source isSNP SNP00021346 consequence FN: 522678CB1.1 319 Missense 239-239 R>H

Allele FN:522678CB1 318 868 868 A>G source isSNP SNP00002987 consequence FN: 522678CB1.1 319 Missense 279-279 Q>R

Allele FN:522678CB1 318 1604 1604 A>G source isSNP SNP00021347 consequence FN: 522678CB1.1 319 Silent 524-524

Allele FN:522678CB1 318 1853 1853 OT source isSNP SNP00002988 consequence FN: 522678CB1.1 319 Silent 607-607 G

Allele FN:522678CB1 318 2159 2159 A>G source isSNP SNP00062663 TABLE 1 (Cont.) consequence FN: 522678CB1.1 319 3' Allele FN:522678CB1 318 2302 2302 A>G source isSNP SNP00021348 consequence FN: 522678CB1.1 319 GIF MMP9-cdna-fwd.gif Link : MMP9_link_genomic

Subsequence GB:HUMIVCOL01 1 764 #320

Subsequencie GB:HUMIVCOL02 865 1117 #321

Subsequence GB:HUMIVCOL03 1218 1386 #322

Subsequence GB:HUMIVCOL04 1487 1635 #323

Subsequenc :e GB:HUMIVCOL05 1736 1929 #324

Subsequence GB:HUMIVCOL06 2030 2223 #325

Subsequence GB:HUMIVCOL07 2324 2520 #326

Subsequenc :e GB:HUMIVCOL08 2621 2796 #327

Subsequenc :e GB:HUMIVCOL09 2897 3196 #328

Subsequence GB:HUMIVCOL10 3297 3456 #329

Subsequenc :e GB:HUMIVC0L11 3557 3727 #330

Subsequenc :e GB:HUMIVCOL12 3828 3951 #331

Subsequenc :e GB:HUMIVCOL13 4052 4371 #332

Subsequence MMP9_cds.l 619 4180 #333

Subsequence MMP9_mrna_build.1 587 4371 #334

CDS MMP9_cds.l 2124 bp 13 exons #333 exon 619 756 exon 875 1107 exon 1228 1376 exon 1497 1625 exon 1746 1919 exon 2040 2213 exon 2334 2510 exon 2631 2786 exon 2907 3186 exon 3307 3446 exon 3567 3717 exon 3838 3941 exon 4062 4180 mRNA MMP9_mrna_build.l 2348 bp 13 exons #334 exon 587 756 exon 875 1107 exon 1228 1376 exon 1497 1625 exon 1746 1919 exon 2040 2213 exon 2334 2510 exon 2631 2786 exon 2907 3186 exon 3307 3446 exon 3567 3717 exon 3838 3941 exon 4061 4371

Allele GB:HUMIVCOL01 320 677 677 A>G source wetSNP GB :HUMIVCOL01.v677. OT consequence MMP9_cds .1 333 Missense 20-20 A:

Allele GB:HUMIVCOL02 321 148 148 A>G source isSNP SNP00101082 consequence MMP9_cds .1 333 Silent 92-92 K TABLE 1 (Cont.)

Allele GB:HUMIVCOL04 323 49 49 A>G source isSNP SNP00101085 consequence MMP9_cds .1 333 Missense 187-187 L>F

Allele GB:HUMIVCOL05 324 48 48 A>G source isSNP SNP00101086 consequence MMP9_cds .1 333 Silent 229-229 A

Allele GB:HUMIVCOL05 324 77 77 A>G source isSNP SNP00021346 consequence MMP9_cds .1 333 Missense 239-239 R>H

Allele GB:HUMIVCOL09 328 252 252 A>G source isSNP SNP00021347 consequence MMP9_cds .1 333 Silent 524-524 I

Allele GB:HUMIVC0L11 330 81 81 G>T source isSNP SNP00002988 consequence MMP9_cds .1 333 Silent 607-607 G

Allele GB :HUMIVCOL13 332 87 87 A>G source wetSNP GB:HUMIVCOL13.v87.OA consequence MMP9_cds .1 333 Silent 694-694 V

Allele GB:HUMIVCOL13 332 132 132 A>G source wetSNP GB :HUMIVCOL13.vl32.OT consequence MMP9_cds.l 333 3'

Allele GB:HUMIVCOL13 332 274 274 A>G source isSNP SNP00021348 consequence MMP9_cds .1 333 3' GIF MMP9-genomic-fwd.gif

MSF

Full name : megakaryocyte stimulating factor

Link : MSF_link_cdna

Subsequence GB:NM_005807 1 5041 #335 CDS GB:NM_005807.1 4215 bp #336

ORF 34 4248

Allele GB:NM_005807 335 1011 1011 A>G source isSNP SNP00064566 consequence GB:NM. .005807.1 3 33366 S Siilleenntt 3 32266--332266 K

Allele GB:NM_005807 335 2650 2650 A>G source isSNP SNP00108532 consequence GB:NM. .005807.1 3 33366 M Miisssseennssee 8 87733--887733 P>S

Allele GB:NM_005807 335 3171 3171 A>G source isSNP SNP00009620 consequence GB:NM. .005807.1 336 Silent 1046-1046

Allele GB:NM_005807 335 4187 4187 A>G source isSNP SNP00061665 consequence GB:NM. .005807.1 3 33366 M Miisssseennssee 1 1338855--11338855 A>V

Allele GB:NM_005807 335 4760 4760 A>G source isSNP SNP00009621 consequence GB :NM. .005807.1 336 GIF MSF-cdna-fwd.gif Link : MSF_link_genomic

Subsequence MSF_cds .1 181003 197905 #337 Subsequence MSF_cds .2 181003 197905 #338 Subsequence MSF_cds .3 181003 197905 #339 Subsequence MSF_cds .4 181003 197905 #340 TABLE 1 (Cont.)

Subsequence GB:AL133553_7 1 214019 #341 Subsequence MSF_mrna_build.l 180982 198681 #342 CDS MSF eds.3 3936 bp 10 exons #339 exon 181003 181078 exon 184218 184340 exon 185719 185838 exon 190445 193267 exon 193920 193997 exon 195161 195297 exon 195567 195723 exon 196302 196499 exon 196896 197021 exon 197808 197905 mRNA MSF_mrna_build.l 5012 bp 12 exons #342 exon 180982 181078 exon 184218 184340 exon 185719 185838 exon 188235 188384 exon 188921 189049 exon 190445 193267 exon 193920 193997 exon 195161 195297 exon 195567 195723 exon 196302 196499 exon 196896 197021 exon 197808 198681

CDS MSF_cds.4 3813 bp 9 exons #340 exon 181003 181078 exon 185719 185838 exon 190445 193267 exon 193920 193997 exon 195161 195297 exon 195567 195723 exon 196302 196499 exon 196896 197021 exon 197808 197905

CDS MSF_cds.l 4215 bp 12 exons #337 exon 181003 181078 exon 184218 184340 exon 185719 185838 exon 188235 188384 exon 188921 189049 exon 190445 193267 exon 193920 193997 exon 195161 195297 exon 195567 195723 exon 196302 196499 exon 196896 197021 exon 197808 197905

CDS MSF_cids.2 4092 bp 11 exons #338 exon 181003 181078 exon 185719 185838 exon 188235 188384 exon 188921 189049 exon 190445 193267 TABLE 1 (Cont.) exon 193920 193997 exon 195161 195297 exon 195567 195723 exon 196302 196499 exon 196896 197021 exon 197808 197905

All( 3le GB:AL133553_ .7 341 190505 19050Ξi G>T source wetSNP GB:AL133553_7.vl90505.A>C consequence MSF_cds .3 339 Missense 127-127 D>A consequence MSF_cds .4 340 Missense 86-86 D>A consequence MSF_cds .1 337 Missense 220-220 D>A consequence MSF_cds .2 338 Missense 179-179 D>A

All.sle GB:AL133553_ .7 341 190559 190559i A>G source wetSNP GB:AL133553_7.vl90559.OT consequence MSF_cds .3 339 Missense 145-145 T>M consequence MSF_cds .4 340 Missense 104-104 T>M consequence MSF_cds .1 337 Missense 238-238 T>M consequence MSF_cds .2 338 Missense 197-197 T>M

All.sle GB:AL133553_ .7 341 190755 190755 ; A>G source wetSNP GB:AL133553_7.V190755.G>A consequence MSF_cds .3 339 Silent 210-210 K consequence MΞF_cds .4 340 Silent 169-169 K consequence MSF_cds .1 337 Silent 303-303 K consequence MSF_cds .2 338 Silent 262-262 K

All(ale GB:AL133553_ .7 341 190824 190824 A>G source isSNP SNP00064566 consequence MSF_cds .3 339 Silent 233-233 R consequence MSF_cds .4 340 Silent 192-192 K consequence MSF_cds .1 337 Silent 326-326 K consequence MSF_cds .2 338 Silent 285-285 ^■ K

All< =le GB:AL133553_ .7 341 19246Ξi 192463 A>G source isSNP SNP00108532 consequence MSF_cds .3 339 Missense 780-780 P>S consequence MSF_cds . 340 Missense 739-739 P>S consequence MSF_cds .1 337 Missense 873-873 P>S consequence MSF_cds .2 338 Missense 832-832 P>S

All< 3le GB:AL133553_ .7 341 192984 192984 A>G source isSNP SNP00009620 consequence MSF_cds .3 339 Silent 953-953 P consequence MSF_cds .4 340 Silent 912-912 P consequence MSF_σds .1 337 Silent 1046-1046 P consequence MSF_cds .2 338 Silent 1005-1005 P

All(sle GB:AL133553_ .7 341 193235 19323Ei A>G source wetSNP GB:ALl33553_7.vl93235.A>G consequence MSF_cds .3 339 Missense 1037-1037 N>S consequence MSF_cds . 340 Missense 996-996 N>S consequence MSF_cds .1 337 Missense 1130-1130 N>S consequence MSF_cds .2 338 Missense 1089-1089 N>S

All,ele GB:AL133553_ .7 341 193258 19325E 1 A>G source wetSNP GB:ALl33553_7.vl93258.A>G consequence MSF_σds .3 339 Missense 1045-1045 M>V consequence MSF_cds . 340 Missense 1004-1004 M>V consequence MSF_cds .1 337 Missense 1138-1138 M>V consequence MSF_cds .2 338 Missense 1097-1097 M>V

All.ele GB:AL133553_ 1 341 1^,669. 196691 G>T TABLE 1 (Cont.) source isSNP SNP01 .023429 consequence MSF_cds .3 339 Intron consequence MSF_cds .4 340 Intron consequence MSF_cds .1 337 Intron consequence MSF_cds .2 338 Intron

Allele GB:AL133553_ .7 341 197844 197844 A>G source isSNP SNP00061665 consequence MSF_cds .3 339 Missense 1292- -1292 A>V consequence MSF_cds .4 340 Missense 1251- -1251 A>V consequence MSF_cds .1 337 Missense 1385- -1385 A>V consequence MSF_cds .2 338 Missense 1344- -1344 A>V

Allele GB:AL133553_ .7 341 198417 198417 A>G source isSNP SNP00009621 consequence MSF_cds .3 339 3' consequence MSF_cds .4 340 3' consequence MSF_cds .1 337 3' consequence MSF_cds .2 338 3'

GIF MSF-genomic-fwd.gif

NC0R2

Full name : nuclear receptor co-repressor 2

Link : NCOR2_link_cdna

Subsequence GB:AF125672 1 8686 #343

CDS GB:AF125672.1 7524 bp #344

ORF 157 7680 Allele GB:AF125672 343 165 165 G>T source isSNP SNP00035702 consequence GB:AF125672.1 344 Silent 3-3 G

Allele GB:AF125672 343 618 618 A>G source isSNP SNP00105557 consequence GB:AF125672.1 344 Silent 154-154

Allele GB:AF125672 343 • 2859 2859 A>G source isSNP SNP00101011 consequence GB:AF125672.1 344 Silent 901-901

Allele GB:AF125672 343 4728 4728 A>G source isSNP SNP00075034 consequence GB:AF125672.1 344 Silent 1524-1524

Allele GB:AF125672 343 4749 4749 A>G source isSNP SNP00069757 consequence GB:AF125672.1 344 Silent 1531-1531

Allele GB:AF125672 343 4957 4957 A>G source isSNP SNP00101012 consequence GB:AF125672.1 344 Missense 1601-1601 Y>H

Allele GB:AF125672 343 5085 5085 A>G source isSNP SNP00075035 consequence GB:AF125672.1 344 Silent 1643-1643 R

Allele GB:AF125672 343 5100 5100 A>G source isSNP SNP00075036 consequence GB:AF125672.1 344 Silent 1648-1648 N

Allele GB:AF125672 343 5221 5221 A>G source isSNP SNP00012485 consequence GB-.AF125672.1 344 Missense 1689-1689 T>A

Allele GB:AF125672 343 7405 7405 A>G TABLE 1 (Cont.) source isSNP SNP00015859 consequence GB:AF125672.1 344 Missense 2417-2417 P>S

Allele GB:AF125672 343 7431 7431 A>G source isSNP SNP00101013 consequence GB:AF125672.1 344 Silent 2425-2425

Allele GB:AF125672 343 7751 7751 A>G source isSNP SNP00101014 consequence GB:AF125672.1 344 3'

Allele GB:AF125672 343 8597 8597 A>G source isSNP SNP00062569 consequence GB:AF125672.1 344 3'

Allele GB:AF125672 343 8602 8602 A>G source isSNP SNP00012487 consequence GB:AF125672.1 344

GIF NCOR2-cdna-fwd.gif

NOG

Full name : NOG

Link : NOG_link_genomic

Subsequence GB:AC005553 1 179651 #345 Subsequence NOG_cds .1 146202 145504 #346 Subsequence NOG_mrna_build.l 147012 145466 #347

CDS NOG_cds.l 699 bp 1 exon #346 exon 146202 145504 mRNA NOG_mrna_build.l 1547 bp 1 exon #347 exon 147012 145466 Allele GB:AC005553 345 145585 145585 A>G source wetSNP GB:AC005553.vl45585.G>A consequence NOG_cds .1 346 Silent 206-206 GIF NOG-genomic-rev.gif

NOTCH3

Link : NOTCH3 link cdna

Subsequenc :e GB:NOTCH3 1 8091 #348

CDS GB:NOTCH3.1 6966 bp #349

ORF 79 7044

Allele GB-.NOTCH3 348 1218 1218 A>G source isSNP SNP00116668 consequence GB:NOTCH3.1 349 Silent 380-380

Allele GB:NOTCH3 348 1565 1565 A>G ^• source isSNP SNP00116669 consequence GB:NOTCH3.1 349 Missense 496-496 P>L

Allele GB:NOTCH3 348 2616 2616 A>G source isSNP SNP00116670 consequence GB:NOTCH3.1 349 Silent 846-846

Allele GB:NOTCH3 348 4520 4520 A>G source isSNP SNP00116671 consequence GB:NOTCH3.1 349 Missense 1481-1481 D>G

Allele GB:NOTCH3 348 5740 5740 A>G source isSNP SNP00054178 consequence GB:NOTCH3.1 349 Missense 1888-1888 F>L TABLE 1 (Cont.)

Allele GB:NOTCH3 348 6355 6355 A>G source isSNP SNP00037780 consequence GB:NOTCH3.1 349 Missense 2093-2093 A>T Allele GB:NOTCH3 348 6516 6516 A>G source isSNP SNP00054179 consequence GB:NOTCH3.1 349 Silent 2146-2146 Allele GB:NOTCH3 348 6746 6746 A>G source isSNP SNP00048081 consequence GB:NOTCH3.1 349 Missense 2223-2223 V>A Allele GB:NOTCH3 348 7733 7733 A>G source isSNP SNP00037781 consequence GB:NOTCH3.1 349 3' Allele GB:NOTCH3 348 7881 7881 A>G source isSNP SNP00062225 consequence GB:NOTCH3.1 349 3' Allele GB:NOTCH3 348 7914 7914 A>G source isSNP SNP00066446 consequence GB:NOTCH3.1 349 3' Allele GB:NOTCH3 348 8023 8023 source isSNP SNP00066447 consequence GB:NOTCH3.1 349 GIF NOTCH3-cdna-fwd.gif Link : NOTCH3_link_genomic

Subsequence NOTCH3_cds .1 40735 3819 #350 Subsequence GB:AC004663_1 1 41150 #351 CDS NOTCH3_cds.l 6846 bp 32 exons #350 exon 40733 40657 exon 35676 35534 exon 35455 35117 exon 35024 34902 exon 34814 34581 exon 32585 32430 exon 32331 32146 exon 31505 31392 exon 31151 31038 exon 30495 30262 exon 30145 30035 exon 28836 28644 exon 28565 28414 exon 28176 28063 exon 27607 27452 exon 24958 24733 exon 24319 24118 exon 23985 23838 exon 23413 23229 exon 22653 22521 exon 22439 22182 exon 22098 21980 exon 2 211224477 20682 exon 1 177555577 17225 exon 13982 13828 exon 13710 13488 exon 13327 13243 exon 10568 10406 exon 9248 8944 TABLE 1 (Cont.) exon 8672 8525 exon 5719 5622 exon 4871 3819 Allele GB:AC004663. 1 351 3796 3796 A>T source wetSNP GB:AC004663_l.v3796.A>T consequence NOTCH3_cds .1 350 3'

Allele GB:AC004663_{. .}1 351 4117 4117 A>G source isSNP SNP00048081 consequence NOTCH3_cds .1 350 Missense 2183-2183 A>V

Allele GB:AC004663..1 351 4347 4347 A>G source isSNP SNP00054179 consequence NOTCH3_cds .1 350 Silent 2106-2106

Allele GB:AC004663._.1 351 4508 4508 A>G source isSNP SNP00037780 consequence N0TCH3_cds .1 350 Missense 2053-2053 A>T

Allele GB:AC004663._.1 351 5727 5727 A>G source wetSNP GB:AC004663_l.v5727.A>G consequence N0TCH3_cds.l 350 Intron

Allele GB:AC004663_.1 351 5943 5943 A>G source dbSNP gnl|dbSNP|ss730238_allele consequence NOTCH3_cds .1 350 Intron

Allele GB:AC004663_1 351 17519 17519 A>G source isSNP SNP00116671 consequence NOTCH3_cds .1 350 Missense 1441-1441 D>G

Allele GB:AC004663_ 1 351 22353 22353 A>G source wetSNP GB:AC004663_l.v22353. T consequence NOTCH3_cds 1 350 Missense 1143-1143 V>M

Allele GB:AC004663_.1 351 23922 23922 OG source wetSNP GB:AC004663_l.v23922.OG consequence NOTCH3_cds 1 350 Missense 980-980 A>P

Allele GB:AC004663__.1 351 24045 24045 A>G source wetSNP GB:AC004663 l.v24045.T>C consequence N0TCH3_cds.l 350 Intron

Allele GB:AC004663_.1 351 27480 27480 A>G source isSNP SNP00116670 consequence NOTCH3_cds.l 350 Silent 806-806

Allele GB:AC004663_.1 351 28173 28173 A>G source wetSNP GB : AC004663_l .v28173 . OT consequence NOTCH3_σds 1 350 Missense 727-727 R>H

Allele GB:AC004663_.1 351 28749 28749 A>G source wetSNP GB : AC004663_l .v28749 . OT consequence NOTCH3_cds 1 350 Missense 640-640 R>H

Allele GB:AC004663_.1 351 29997 29997 OG source wetSNP GB : AC004663_l .v29997 . G>C consequence N0TCH3_cds 1 350 Intron

Allele GB:AC004663_ _.1 351 32482 32482 A>G source isSNP SNP00116668 consequence NOTCH3_cds . l 350 Silent 340-340

GIF NOTCH3 -genomic-rev . gif TABLE 1 (Cont.)

NPR2

Full name : Atrionatriuretic Peptide Receptor Type B

Link : NPR2_link_σdna

Subsequence GB:HUMGUANCYC 1 4081 #352 CDS GB:HUMGUANCYC.2 3144 bp #353

ORF 651 3794

Allele GB :HUMGUANCYC 352 2222 2222 A>G source isSNP SNP00028343 consequence GB:HUMGUANCYC .2 353 Silent 524-524

GIF NPR2-cdna-fwd.gif

OGN

Full name : osteoglycin

Link : OGN_link_cdna

Subsequence GB:HSM801395 2101 #354 CDS GB:HSM801395.1 441 bp #355

ORF 1 441 Allele GB:HSM801395 354 64 64 A>G source isSNP SNP00100803 consequence GB:HSM801395.1 355 Missense 22-22 L>F Allele GB:HSM801395 354 909 909 A>G source isSNP SNP00011097 consequence GB:HSM801395.1 355 GIF OGN-cdna-fwd.gif Link : OGN_link_genomic

Subsequence OGN_cds .2 48897 32003 #356 Subsequence GB:AL354924_2 1 192427 #357 Subsequence OGN_mrna_build.2 50083 30350 #358 mRNA OGN_mrna_build.2 2726 bp 7 exons #358 exon 50083 49983 exon 48969 48721 exon 46672 46579 exon 38619 38461 exon 35431 35229 exon 32679 32584 exon 32173 30350

CDS OGN_cids.2 900 bp 6 exons #356 exon 48897 48721 exon 46672 46579 exon 38619 38461 exon 35431 35229 exon 32679 32584 exon 32173 32003

Allele GB : AL 154924_2 357 31535 31535 A>G source isSNP SNP00011097 consequence OGN__σds .2 356 3' Allele GB:AL354924_2 357 35339 35339 A>G source isSNP SNP00100803 consequence OGN_cds.2 356 Missense 175-175 L>F GIF OGN-genomic-rev.gif TABLE 1 (Cont.)

OMD

Full name : osteomodulin

Link : OMD_link_cdna

Subsequence GB:OMD 2263 #359 CDS GB:OMD.l 1266 bp #360

ORF 101 1366 Allele GB:OMD 359 159 159 OG source isSNP SNP00023658 consequence GB:OMD.l 360 Missense 20-20 OS

Allele GB : OMD 359 762 762 A>G source isSNP SNP00023659 consequence GB:OMD.l 360 Missense 221-221 S>N

Allele GB : OMD 359 1969 1969 A>G source isSNP SNP00023660 consequence GB:OMD.l 360 3'

Allele GB:OMD 359 2071 2071 G>T source isSNP SNP00106046 consequence GB:OMD.l 360 GIF OMD-cdna-fwd.gif Link : FL_1258977_link_genomic

Subsequence GB:AB009589 1 12414 #361 Subsequence GB:AB009589_1258977CD1 8540 10946 #362 Subsequence FL_1258977_mrna_build.1 1685 11855 #363 mRNA FL_1258977_mrna_build.l 2396 bp 3 exons #363 exon 1685 1892 exon 8524 9479 exon 10624 11855 CDS GB:AB009589_1258977CD1 1263 bp 2 exons #362 exon 8540 9479 exon 10624 10946 Allele GB:AB009589 361 8598 8598 OG source isSNP SNP00023658 consequence GB:AB009589_1258977CD1 362 Missense 20-20 OS

Allele GB:AB009589 361 9201 9201 A>G source isSNP SNP00023659 consequence GB:AB009589 1258977CD1 362 Missense 221-221

S>N Allele GB:AB009589 361 10042 10042 A>G source dbS^*NP gnl|dbSNP|ss312223_allele consequence GB:AB009589_1258977CD1 362 Intron

Allele GB:AB009589 361 10596 10596 A>G source wetSNP GB:AB009589.vl0596.A>G consequence GB:AB009589_1258977CD1 362 Intron

Allele GB:AB009589 361 11552 11552 A>G source isSNP SNP00023660 consequence GB:AB009589_1258977CD1 ^• 362

Allele GB:AB009589 361 11654 11654 OT source isSNP SNP00106046 consequence GB:AB009589_1258977CD1 362 GIF OMD-genomic-fwd.gif

PDCD6IP Full name programmed cell death 6-interacting protein TABLE 1 (Cont.)

Link : PDCD6IP_link_cdna

Subsequence GB:AF151793 1 3221 #364

CDS GB:AF151793.1 2607 bp #365

ORF 127 2733 Allele GB:AF151793 364 1051 1051 A>G source isSNP SNP00029958 consequence GB:AF151793.1 365 Missense 309-309 T>A

Allele GB:AF151793 364 1258 1258 A>G source isSNP SNP00108790 consequence GB:AF151793.1 365 Missense 378-378 V>I

Allele GB:AF151793 364 1298 1298 G>T source isSNP SNP00108791 consequence GB:AF151793.1 365 Missense 391-391 L>W

Allele GB:AF151793 364 1695 1695 A>G source isSNP SNP00093444 consequence GB:AF151793.1 365 Silent 523-523

Allele GB:AF151793 364 2230 2230 A>G source isSNP SNP00121559 consequence GB:AF151793.1 365 Missense 702-702 R>G

Allele GB:AF151793 364 2315 2315 A>G source isSNP SNP00006604 consequence GB:AF151793.1 365 Missense 730-730 L>S

Allele GB:AF151793 364 2386 2386 A>G source isSNP SNP00029960 consequence GB:AF151793.1 365 Missense 754-754 P>S

Allele GB:AF151793 364 2421 2421 A>G source isSNP SNP00121560 consequence GB:AF151793.1 365 Silent 765-765

GIF PDCD6IP-cdna-fwd.gif

PDNP1

Full name : phosphodiesterase I (nucleotide pyrophosphatase I (homologous to mouse Ly-41 antigen) )

Link : PDNPl_link_cdna

Subsequence EM:HSAUTOTAX 1 3231 #366

CDS EM:HSAUTOTAX.2 2748 bp #367

ORF 50 2797 Allele EM:HSAUTOTAX 366 342 342 A>G source isSNP SNP00025434 consequence EM:HSAUTOTA .2 367 Missense 98-98 A A>V

Allele EM:HSAUTOTAX 366 696 696 A>G source isSNP SNP00075872 consequence EM:HSAUTOTAX.2 367 Missense 216-216 T>I

Allele EM:HSAUTOTAX 366 1682 1682 A>G source isSNP SNP00025435 consequence EM:HSAUTOTAX.2 367 Missense 545-545 P>S

Allele EM:HSAUTOTAX 366 1789 1789 A>G source isSNP SNP00004604 consequence EM:HSAUTOTAX.2 367 Silent 580-580 H

Allele EM:HSAUTOTAX 366 2398 2398 G>T source isSNP SNP00122211 consequence EM:HSAUTOTAX.2 367 Silent 783-783 V

Allele EM:HSAUTOTAX 366 2539 2539 A>G TABLE 1 (Cont.) source isSNP SNP00004605 consequence EM:HSAUTOTAX.2 367 Silent 830-830 Allele EM:HSAUTOTAX 366 2681 2681 G>T source isSNP SNP00059344 consequence EM:HSAUTOTA .2 367 Silent 878-878 GIF PDNPl-cdna-fwd.gif Link : PDNPl_link_genomic

Subsequence IN: 98092911313498 4217 4948 #368 Subsequence IN: 98061109562226435 5050 5980 #369 Subsequence IN: 98092910591328158 3611 4115 #370 Subsequence IN: 98092911013628201 100 699 #371 Subsequence IN: 98092911024828217 2027 2526 #372 Subsequence IN: 98092911044928261 3068 3509 #373 Subsequence IN: 98092911065328292 801 1418 #374 Subsequence IN: 98092913141116289 6183 6572 #375 Subsequence IN: 98111010592914993 1520 1926 #376 Subsequence IN: 98111011021915028 2628 2967 #377 Allele IN: 98092910591328158 370 232 232 A>G source isSNP SNP00025435 Allele IN:98092913141116289 375 189 189 G>T source isSNP SNP00059344

PLA2G2A

Full name : phospholipase A2 , group IIA

Link : PLA2G2A_link_cdna

Subsequence GB:HUMRASFAB 1 854 #378 CDS GB:HUMRASFAB.1 435 bp #379

ORF 136 570 Allele GB:HUMRASFAB 378 267 267 A>G source isSNP SNP00010003 consequence GB :HUMRASFAB .1 379 Silent 44-44 Y Allele GB:HUMRASFAB 378 800 800 A>G source isSNP SNP00021612 consequence GB :HUMRASFAB .1 379 GIF PLA2G2A-cdna-fwd.gif Link : PLA2G2A_link_genomic

Subsequence PLA2G2A_cds .1 51704 48629 #380 Subsequence PLA2G2A_.mrna._buiId.1 52537 48418 #381 Subsequence GB:AL358253_1 1 180550 #382 Subsequence LG: 474322.13_mrna_build.1 52786 48418 #383

Subsequence PLA2G2A_cds.2 51704 50985 #384 mRNA LG: 474322.13_mrna_build.1 1028 bp 5 exons #383 exon 52786 52511 exon 51810 51665 exon 51455 51311 exon 51052 50946 exon 48771 48418 CDS PLA2G2A_cds .1 435 bp 4 exons #380 exon 51704' 5166"5^" exon 51455 51311 exon 51052 50946 exon 48771 48629 CDS PLA2G2A_cds .2 108 bp 2 exons #384 TABLE 1 (Cont.) exon 51704 51665 exon 51052 50985 mRNA PLA2G2A_mrna_build.l 779 bt > 5 exons #381 exon 52537 52511 exon 51810 51665 exon 51455 51311 exon 51052 50946 exon 48771 48418

Allele GB:AL358253_1 382 51364 51364 A>G source isSNP SNP00010003 consequence PLA2G2A_cds .1 380 Silent 44-44 Y consequence PLA2G2A_cds .2 384 Intron

Allele GB:AL358253_1 382 52584 52584 OG source isSNP SNP00021611 consequence PLA2G2A_cds .1 380 5' consequence PLA2G2A_cds .2 384 5'

GIF PLA2G2A-genomic-rev.gif

PPP1R5

Full name : protein phosphatase 1, regulatory (inhibitor) subunit 5

Link : PPPlR5_link_cdna

Subsequence GB:Y18207_1 1 1158 #385

CDS GB:Y18207 1.1 954 bp #386

ORF 92 1045 Allele GB:Y18207_1 385 571 571 A>G source isSNP SNP00041149 consequence GB:Y18207_1.1 386 Silent 160-160

Allele GB:Y18207_1 385 1096 1096 OT source isSNP SNP00060710 consequence GB:Y18207_1.1 386 GIF PPPlR5-cdna-fwd.gif Link : PPPlR5_link_genomiσ

Subsequence GB:AC020691_2 1 152048 #387 Subsequence PPPlR5_mrna_build .1 103997 10724Ei # #388 Subsequence PPPlR5_cds .1 106194 107132 #389 CDS PPPlR5_σds.l 939 bp 1 exon #389 exon 106194 107132 mRNA PPPlR5_mrna_build.1 1160 bp 2 exons #388 exon 103997 104103 exon 106193 107245

Allele GB:AC020691_2 387 106523 106523 G>T source wetSNP GB:AC020691_ 2.vl06523.T>G consequence PPPlR5_cds . 1 389 Missense 110-110 D>E

Allele GB:AC020691_2 387 106658 106658 A>G source isSNP SNP0C 1041149 consequence PPPlR5_cds . 1 389 Silent 155-155

Allele GB:AC020691_2 387 107183 107183 G>T source isSNP SNP00060710 consequence PPPlR5_cds .1 389 GIF PPPlR5-genomic-fwd.gif TABLE 1 (Cont.)

PRELP

Full name : proline arginine-rich end leucine-rich repeat protein

Link : PRELP_link_cdna

Subsequence GB:HSU29089 1 1560 #390

CDS GB:HSU29089.1 1149 bp #391

ORF 129 1277 Allele GB:HSU29089 390 1170 1170 G>T source isSNP SNP00001359 consequence GB:HSU29089.1 391 Missense 348-348 N>H

Allele GB:HSU29089 390 1489 1489 OT source isSNP SNP00001361 consequence GB:HSU29089.1 391 GIF PRELP-cdna-fwd.gif Link : PRELP_link_genomic

Subsequence PRELP_cds.l 82496 86192 #392 Subsequence GB:AC022000_1 1 154681 #393 Subsequence PRELP_mrna_b ild.1 75139 86474 #394 CDS PRELP_σds.l 1149 bp 2 exons #392 exon 82496 83468 exon 86017 86192 mRNA PRELP_mrna_build.l 1559 bp 3 exons #394 exon 75139 75250 exon 82480 83468 exon 86017 86474 Allele GB:AC022000_1 393 86085 86085 G>T source isSNP SNP00001359 consequence PRELP_cds .1 392 Missense 348-348 N>H Allele GB: C022000_1 393 86404 86404 G>T source isSNP SNP00001361 consequence PRELP_cds .1 392 3' GIF PRELP-genomic-fwd.gif

PRSS11

Full name : serine protease .

Link : FL_1787335_link_cdna

Subsequence FN: 1787335CB1 2054 #395 CDS FN:1787335CB1.1 1443 bp #396

ORF 49 1491 Allele FN:1787335CB1 395 150 150 A>G source isSNP SNP00068999 consequence FN: 1787335CB1.1 396 Silent 34-34 A

Allele FN:1787335CB1 395 156 156 G>T source isSNP SNP00117078 consequence FN: 1787335CB1.1 396 Silent 36-36 G

Allele FN:1787335CB1 395 914 914 A>G source isSNP SNP00120314 consequence FN: 1787335CB1.1 396 Missense 289-289 Q>R

Allele FN:1787335CB1 395 1321 1321 OG source isSNP SNP00105589 consequence FN: 1787335CB1.1 396 Missense 425-425 A>P

Allele FN:1787335CB1 395 1521 1521 A>G source isSNP SNP00105590 consequence FN: 1787335CB1.1 396 TABLE 1 (Cont.)

GIF PRSSll-cdna-fwd.gif Link : FL_1787335_link_genomic

Subsequence GB:AF157623_1_1787335CD1 17526 70213 #397 Subsequence GB:AF157623_1 1 79597 #398 Subsequence FL_1787335_mrna_build.1 17478 70761 #399 CDS GB:AF157623_1_1787335CD1 1443 bp 9 exons #397 exon 17526 17997 exon 44770 44869 exon 45290 45494 exon 62561 62755 exon 63240 63272 exon 64526 64640 exon 65966 66023 exon 67827 67922 exon 70045 70213 mRNA FL_1787335_mrna_build.l 2039 bp 9 exons #399 exon 17478 17997 exon 44770 44869 exon 45290 45494 exon 62561 62755 exon 63240 63272 exon 64526 64640 exon 65966 66023 exon 67827 67922 exon 70045 70761

Allele GB:AF157623_1 398 17627 17627 A>G source isSNP SNP00068999 consequence GB:AF157623_1_1787335CD1 397 Silent

34-34 A Allele GB:AF157623_1 398 17633 17633 G>T source isSNP SNP00117078 consequence GB:AFl57623_l_1787335CDl 397 Silent

36-36 G Allele GB:AF157623_1 398 21721 21721 A>G source isSNP SNP00101582 consequence GB:AF157623_1_1787335CD1 397 Intron

Allele GB:AF157623_1 398 35790 35790 A>G source isSNP SNP00049308 consequence GB:AF157623_1_1787335CD1 397 Intron

Allele GB:AF157623_1 398 44762 44762 G>T source wetSNP GB:AF157623_ .l.v44762.G>T consequence GB: F157623_1_1787335CD1 397 Intron

Allele GB:AF157623_1 398 45470 45470 A>G source wetSNP GB:AF157623_ 1.V45470.OT consequence GB:AF157623_1_1787335CD1 397 Silent

251-251 I Allele GB:AF157623_1 398 45587 45587 A>G source wetSNP GB:AF157623_ .1.V45587.0T consequence GB:AF157623_1_1787335CD1 397 Intron

Allele GB:AF157623_1 398 47792 47792 A>G source isSNP SNP00105588 consequence GB :AF157623_1_1787335CD1 397 Intron

Allele GB:AF157623_1 398 47834 47834 A>G source isSNP SNP00120312 consequence GB:AF157623_l_1787335CDl 397 Intron TABLE 1 (Cont.)

Allele GB:AF157623_ 1 398 47913 47913 A>G source isSNP SNP00120313 consequence GB:AF157623_1_1787335CD1 397 Intron

Allele GB:AF157623_1 398 62541 62541 A>G source wetSNP GB:AF157623_l.v62541.G>A consequence GB:AF157623_1_1787335CD1. 397 Intron

Allele GB:AF157623_1 398 62545 62545 A>G source wetSNP GB:AF157623_l.v62545.G>A consequence GB:AF157623_1_1787335CD1 397 Intron

Allele GB:AF157623_1 398 62649 62649 A>G source isSNP SNP00120314 consequence GB:AF157623_1_1787335CD1 397 Missense

289-289 Q>R Allele GB:AF157623_ ,1 398 63355 63360 TGTTTT>TT source wetSNP GB:AF157623_l.v63355.TGTTTT>TT consequence GB:AF157623_1_1787335CD1 397 Intron

Allele GB:AF157623_ 1 398 70243 70243 A>G source isSNP SNP00105590 consequence GB:AF157623_1_1787335CD1 397 3'

GIF PRSSll-genomic-fwd .gif

PTGS2

Full name : Prostaglandin-endoperoxide Synthase 2

Link : PTGS2 link cdna

Subsequenc :e EM:HSCYCLOX 1 3387 #400

Allele EM:HSCYCLOX 400 403 403 OG source isSNP SNP00046167

Allele EM:HSCYCLOX 400 880 880 OT source isSNP SNP00076329

Allele EM:HSCYCLOX 400 2033 2033 A>G source isSNP SNP00076330

Allele EM:HΞCYCLOX 400 2300 2300 A>G source isSNP SNP00046168

Allele EM:HSCYCLOX 400 2983 2983 A>G source isSNP SNP00046169

.nk : PTGS2 :_link_genomic

Subsequence GB:HUMPTGS2 101 11097 #401

Subsequence PTGS2. _cds.1 1925 8146 #402

Subsequenc :e PTGS2. _mrna_build.1 1828 9607 #403

CDS PTGS2. _cds .1 1815 bp 10 exons #402 exon 1925 1976 exon 2777 2893 exon 3014 3157 exon 3811 3954 exon 4670 4851 exon 5584 5667 exon 5787 6033 exon 6315 6601 exon 7103 7250 exon 7737 8146 mRNA PTGS2_ _mrna_build.1 3373 ]bp 10 exons #403 exon 1828 1976 exon 2777 2893 TABLE 1 (Cont.) exon 3014 3157 exon 3811 3954 exon 4670 4851 exon 5584 5667 exon 5787 6033 exon 6315 6601 exon 7103 7250 exon 7737 9607

Allele GB:HUMPTGS2 401 3050 3050 OG source wetSNP GB :HUMPTGS2.v3050 .G>C consequence PTGS2_cds .1 402 Silent 102- -102 V

Allele GB:HUMPTGS2 401 3090 3090 A>G source wetSNP GB :HUMPTGS2.v3090 .OT consequence PTGS2_cds.1 402 Intron

Allele GB:HUMPTGS2 401 3174 3174 OG source wetSNP GB :HUMPTGS2. 3174 .G>C consequence PTGS2_cds .1 402 Intron

Allele GB:HUMPTGS2 401 3793 3793 A>G source wetSNP GB :HUMPTGS2.v3793 .OT consequence PTGS2_cds .1 402 Silent 132- -132

Allele GB:HUMPTGS2 401 3829 3829 A>G source wetSNP GB :HUMPTGS2.v3829 .T>C consequence PTGS2_cds .1 402 Silent 144- -144

Allele GB:HUMPTGS2 401 5605 5605 A>G source wetSNP GB :HUMPTGS2.v5605 .G>A consequence PTGS2_cds .1 402 Intron

Allele GB :HUMPTGS2 401 5676 5681 TATTTT>TT source wetSNP GB:HUMPTGS2.v5676 .TATTTT>TT consequence PTGS2_cds .1 402 Intron

Allele GB :HUMPTGS2 401 5746 5746 G>T source isSNP SNP00076329 consequence PTGS2_cds .1 402 Stop 261-261

Allele GB :HUMPTGS2 401 6249 6249 A>G source wetSNP GB:HUMPTGS2.v6249 , .OA consequence PTGS2_cds .1 402 Silent 335- -335 V

Allele GB:HUMPTGS2 401 6444 6444 A>G source wetSNP GB :HUMPTGS2.v6444 .G>A consequence PTGS2_cds .1 402 Silent 400- -400

Allele GB:HUMPTGS2 401 6453 6453 A>G source wetSNP GB :HUMPTGS2.v6453 .T>C consequence PTGS2_cds .1 402 Silent 403- -403

Allele GB :HUMPTGS2 401 7581 7581 A>G source wetSNP GB :HUMPTGS2. 7581 .T>C consequence PTGS2_cds .1 402 Intron

Allele GB:HUMPTGS2 401 7763 7763 A>G source wetSNP GB :HUMPTGS2.v7763 .T>C consequence PTGS2_σds .1 402 Missense 511- -511 V>A

Allele GB:HUMPTGS2 401 7986 7986 G>T source wetSNP GB :HUMPTGS2.v7986 .OA consequence PTGS2_σds .1 402 Silent 585- -585 R

Allele GB:HUMPTGS2 401 8167 8167 A>G source isSNP SNP00076330 consequence PTGS2_cds .1 402 3'

Allele GB:HUMPTGS2 401 8434 8434 A>G source isSNP SNP00046168 TABLE 1 (Cont.) consequence PTGS2. .cds.l 402 3'

Allele GB:HUMPTGS2 401 8473 8473 A>G source isSNP SNP00012871 consequence PTGS2. .cds.l 402 3'

Allele GB:HUMPTGS2 401 9102 9102 A>G source isSNP SNP00046169 consequence PTGS2. .cds.l 402 3'

GIF PTGS2-genomic-fwd.gif

PTHLH

Full name : PTHLH

Link : PTHLH_link_genomic

Subsequence PTHLH_cds .1 106964 117899 #404 Subsequence GB:AC008011_6 1 183178 #405 Subsequence PTHLH_mrna_build.1 106942 118367 #406 CDS PTHLH_cds.l 534 bp 3 exons #404 exon 106964 107064 exon 112688 113110 . exon 117890 117899 mRNA PTHLH_mrna_build.l 1024 bp 3 exons #406 exon 106942 107064 exon 112688 113110 exon 117890 118367 Allele GB :AC008011_6 405 113450 113450 A>G source isSNP SNP00043978 consequence PTHLH_cds .1 404 ^' Intron

Allele GB:AC008011_6 405 115075 115075 A>G source dbSNP gnl | dbSNP| ssl455356_allele consequence PTHLH_cds .1 404 Intron

Allele GB:AC008011_6 405 115160 115160 A>G source dbSNP gnl | dbSNP| ssl067559_allele consequence PTHLH_cds.l 404 Intron

GIF PTHLH-genomic-fwd.gif

PTHR1

Full name : PTHR1

Link : PTHRl_link_cdna

Subsequence GB :HUMPTHR 1 1948 #407 CDS GB:HUMPTHR.1 1782 bp #408

ORF 29 1810

Allele GB:HUMPTHR 407 1417 1417 A>G source isSNP SNP000 07059 consequence GB:HUMPTHR.1 408 Silent 463-463 N

GIF PTHR1- -cdna-fwd.gif

Link : : PTHR1 _1ink_genomic

Subsequence GB:HSPTHPRH1 1 262 #409 Subsequence GB:HSPTHPRH2 363 769 #410 Subsequence GB:HSPTHPRH3 870 1168 #411 Subsequence GB:HSPTHPRH4 1269 2146 #412 Subsequence GB:HSPTHPRH5 2247 3249 #413 Subsequence GB:HSPTHPRH6 3350 4062 #414 TABLE 1 (Cont.)

Subsequence GB:HSPTHPRH7 4163 4475 #415

Subsequence GB:HSPTHPRH8 4576 4995 #416

Subsequence GB:HSPTHPRH9 5096 5696 #417

Subsequence PTHRl_cds.l 107 5558 #418

Subsequence PTHRl_mrna_build.. 79 5696 #419

CDS PTHRl. _cds .1 1782 bp 14 ex ns #418 exon 107 181 exon 456 558 exon 936 1070 exon 1436 1546 exon 1655 1773 exon 1959 2053 exon 2351 2546 exon 2980 3133 exon 3547 3607 exon 3938 4004 exon 4273 4367 exon 4628 4769 exon 4851 4892 exon 5172 5558 mRNA PTHRl. _mrna_bui1d.1 1948 bp 14 exons #419 exon 79 181 exon 456 558 exon 936 1070 exon 1436 1546 exon 1655 1773 exon 1959 2053 exon 2351 2546 exon 2980 3133 exon 3547 3607 exon 3938 4004 exon 4273 4367 exon 4628 4769 exon 4851 4892 exon 5172 5696

Allele GB:HSPTHPRH3 411 104 104 A>G source wetSNP GB :HSPTHPRH3. l04. G>A consequence PTHRl_cds .1 418 Silent 72-72 A

Allele GB:HSPTHPRH8 416 311 311 A>G source wetSNP GB:HSPTHPRH8.v311.T>C consequence PTHRl_cds .1 418 Silent 463-463 N

GIF PTHRl- -genomic-fwd.gif

RARA

Full name : retinoic acid receptor, alpha

Link : RARA_link_cdna

Subsequence GB:NM_000964 1 2907 #420 CDS GB:NM_000964.1 1389 bp #421

ORF 103 1491 Allele GB:NM_000964 420 2327 2327 A>G source isSNP SNP00016145 consequence GB:NM_000964.1 421 3'

Allele GB:NM 000964 420 2439 2439 A>G TABLE 1 (Cont.) source isSNP SNP00049381 consequence GB:NM_000964.1 421 3' GIF RARA-cdna-fwd.gif

RIN1

Full name : ras inhibitor

Link : RINl_link_cdna

Subsequence GB:HUMRASINF 1 1285 #422

Allele GB:HUMRASINF 422 260 260 A>G source isSNP SNP00123606

Allele GB:HUMRASINF 422 424 424 A>G source isSNP SNP00123607

Allele GB:HUMRASINF 422 722 722 A>G source isSNP SNP00033587

Allele GB:HUMRASINF 422 921 921 A>G source isSNP SNP00007808

ROR2

Full name : receptor tyrosine kinase-like orphan receptor 2

Link : ROR2_link_cdna

Subsequence GB:NM_004560 1 4092 #423 CDS GB:NM_004560.1 2832 bp #424

ORF 200 3031 Allele GB:NM_004560 423 932 932 A>G source isSNP SNP00098926 consequence GB:NM_004560.1 4 42244 M Miisssseennssee 2 24455-- -224455 A>T Allele GB:NM_004560 423 1460 1460 A>G source isSNP SNP00098927 consequence GB:NM_004560.1 4 42244 M Miisssseennssee 4 42211-- -442211 L>F Allele GB:NM_004560 423 1973 1973 A>G source isSNP SNP00098928 consequence GB:NM_004560.1 4 42244 M Miisssseennssee 5 59922-- -559922 F>L Allele GB:NM_004560 423 2287 2287 A>G source isSNP SNP00028168 consequence GB:NM_004560.1 424 Silent 696- -696 Allele GB:NM_004560 423 2353 2353 A>G source isSNP SNP00098929 consequence GB:NM_004560.1 424 Silent 718- -718 Allele GB:NM_004560 423 2654 2654 A>G source isSNP SNP00028169 consequence GB:NM_004560.1 4 42244 M Miisssseennssee 8 81199-- -881199 V>I Allele GB:NM_004560 423 3743 3743 A>G source isSNP SNP00028170 consequence GB:NM_004560.1 424 3' Allele GB:NM_004560 423 3872 3872 G>T source isSNP SNP00074568 consequence GB:NM_004560.1 424 3' Allele GB:NM_004560 423 3919 3919 OT source isSNP SNP00074569 consequence GB:NM_004560.1 424

GIF ROR2-cdna-fwd.gif TABLE 1 (Cont.)

RORA

Full name : RAR-related orphan receptor alpha

Link : RORA_ _link_genomic

Subsequenc :e RORA_cds .1 64220 3076 #425

Subsequenc :e RORA_cds .2 64220 3076 #426

Subsequenc :e RORA_cds .4 64220 3076 #427

Subsequenc :e GB:AC012344_ _4_000018 1 9454 #428

Subsequenc :e GB:AC012344_ _4_000020 9555 21185 #429

Subsequence GB:AC012344_ _4_000021 21286 34347 #430

Subsequenc :e GB:AC012344_ _4_000019 34448 43824 #431

Subsequenc :e GB:AC012344_ _4_000023 43925 65900 #432

Subsequenc :e RORA__mrna_build.1 64309 2885 #433

Subsequenc :e RORA_mrna_b ild.4 64290 2885 #434 mRNA RORA_mrna_build.4 1908 bp 11 exons #434 exon 64290 64084 exon 51847 51714 exon 25290 25205 exon 19553 19412 exon 16417 16022 exon 10425 10304 exon 9288 9156 exon 8488 8381 exon 6690 6580 exon 5625 5513 exon 3240 2885

CDS RORA_σds.l 1671 bp 12 exons #425 exon 64220 64084 exon 43229 43148 exon 41851 41776 exon 25290 25205 exon 19553 19412 exon 16417 16022 exon 10425 10304 exon 9288 9156 exon 8488 8381 exon 6690 6580 exon 5625 5513 exon 3240 3076

CDS RORA_cds.2 1275 bp 11 exons #426 exon 64220 64084 exon 43229 43148 exon 41851 41776 exon 25290 25205 exon 19553 19412 exon 10425 10304 exon 9288 9156 exon 8488 8381 exon 6690 6580 exon 5625 5513 exon 3240 3076 mRNA RORA_mrna_build.1 1951 bp 12 exons #433 exon 64309 64084 exon 43229 43148 TABLE 1 (Cont.) exon 41851 41776 exon 25290 25205 exon 19553 19412 exon 16417 16022 exon 10425 10304 exon 9288 9156 exon 8488 8381 exon 6690 6580 exon 5625 5513 exon 3240 2885

CDS RORA_eds.4 1647 bp 11 exons #427 exon 64220 64084 exon 51847 51714 exon 25290 25205 exon 19553 19412 exon 16417 16022 exon 10425 10304 exon 9288 9156 exon 8488 8381 exon 6690 6580 exon 5625 5513 exon 3240 3076

Alls 3le GB:AC012344_ 4_000020 429 11153 11153 A>G source dbSNP gnl|(ibSNP | ss380580_allele consequence RORA_cds .1 425 Intron consequence RORA_cds .2 426 Intron consequence RORA_cds .4 427 Intron

All<sle GB:AC012344_ _4_000020 429 11182 11182 A>G source dbSNP gnl|ιdbSNP | ss380580_allele consequence RORA_cds .1 425 Intron consequence RORA_cds .2 426 Intron consequence RORA_cds .4 427 Intron

All<sle GB:AC012344_ _4_000020 429 11183 11183 A>T source dbSNP gnl|ιdbSNP| ss507731_allele consequence RORA_cds .1 425 Intron consequence RORA_cds .2 426 Intron consequence RORA_cds .4 427 Intron

All<sle GB:AC012344_ _4_000020 429 11254 11254 A>G source dbSNP gnl|ιdbSNP | ss380580_allele consequence RORA_cds .1 425 Intron consequence RORA_cds .2 426 Intron consequence RORA_cds .4 427 Intron

AILsle GB:AC012344_ _4_000020 429 11255 11255 A>T source dbSNP gnl| dbSNP | ss507731_allele consequence RORA_cds.1 425 Intron consequence RORA_cds .2 426 Intron consequence RORA_cds .4 427 Intron ll.ele GB:AC012344_ _4_000020 429 11264 11264 A>G source dbSNP gnl| dbSNP | ss380580_allele consequence RORA_cds .1 425 Intron consequence RORA_cds .2 426 Intron consequence RORA_σds .4 427 Intron

AILsle GB:AC012344_ _4_000020 429 11265 11265 A>T source dbSNP gnl| dbSNP | ss507731_allele consequence RORA_cds .1 4δ_ Intron TABLE 1 (Cont.) consequence RORA_cds.2 426 Intron consequence RORA_cds .4 427 Intron

Allele GB:AC012344_4_000020 429 11320 11320 A>G source dbSNP gnl | dbSNP | ss380580_allele consequence RORA_cds .1 425 Intron consequence RORA_cds.2 426 Intron consequence RORA_cds .4 427 Intron

GIF RORA-genomic-rev.gif

SCRGl

Full name : scrapie responsive protein

Link : SCRGl_link_genomic

Subsequenc :e SCRGl_cds.l 30577 33650 #435

Subsequence GB:AC009588_4 1 164772 #436

Subsequenc :e SCRGl_mrna_build.1 30561 33845 #437

CDS SCRGl. .eds .1 297 bp 2 exons #435 exon 30577 30818 exon 33596 33650 mRNA SCRGl. _mrna_build.1 508 b; 2 exons #437 exon 30561 30818 exon 33596 33845

GIF SCRGl-genomic-fwd.gif

SCYA20

Full name : small inducible cytokine subfamily A member 20

Link : SCYA20_link_cdna

Subsequence GB:HSU64197 1 821 #438

CDS GB:HSU64197.1 288 bp #439

ORF 43 330 Allele GB:HSU64197 438 341 341 A>G source isSNP SNP00037526 consequence GB:HSU64197.1 439

Allele GB:HSU64197 438 728 728 A>G source isSNP SNP00037527 consequence GB:HSU64197.1 439 GIF SCYA20-cdna-fwd.gif Link : SCYA20_link_genomic

Subsequence SCYA20_cds.l 73925 77096 #440 Subsequence GB:AC027560_2 1 129588 #441 Subsequence SCYA20_mrna_build 1 73883 77577 #442 CDS SCYA20_cds.l 288 bp 4 exons #440 exon 73925 74000 exon 75470 75581 exon 76320 76397 exon 77075 77096 mRNA SCYA20__mrna_build.l 811 bp 4 exons #442 exon 73883 74000 exon 75470 75581 exon 76320 76397 exon 77075 77577 Allele GB:AC027560_2 441 77107 77107 A>G TABLE 1 (Cont.) source isSNP SNP00037526 consequence SCYA20_σds.l 440 3' Allele GB :AC027560_2 441 77493 77493 A>G source isSNP SNP00037527 consequence SCYA20_cds.l 440 3' GIF SCYA20-genomic-fwd.gif

SDC2

Full name : syndecan 2 Link : SDC2_link_cdna Subsecjuence GB:HU1 HSPGC 1 3414 #443 CDS GB :HUMHSPGC .2 1194 bp #444

ORF 1 1194 Allele GB:HUMHSPGC 443 435 435 A>G source isSNP SNP00116695 consequence GB:HUMHSPGC.2 444 Silent 145- -145 Allele GB:HUMHSPGC 443 463 463 OG source isSNP SNP00050825 consequence G GBB ::HHUUMMHHSSPPGGCC ..22 4 44444 M Miisssseennssee 1 15555-- -115555 L>V Allele GB:HUMHSPGC 443 741 741 A>G source isSNP SNP00033651 consequence GB:HUMHSPGC.2 444 Silent 247- -247 Allele GB:HUMHSPGC 443 1041 1041 OT source isSNP SNP00099428 consequence GB:HUMHSPGC.2 444 Silent 347- -347 GIF SDC2-cdna-fwd.gif

SDC4

Full name : syndecan 4

Link : FL_1394592_link_cdna

Subsequence FN: 1394592CB1 1 2112 #445 CDS FN.1394592CB1.1 594 bp #446

ORF 23 616 CDS GB:HS453C12_1394592CD1 594 bp #272

ORF 87967 88026

ORF 100431 100569

ORF 103282 103328

ORF 105787 105985

ORF 108936 109084 mRNA FL_1394592_mrna__build 1 2110 bp #274

ORF 87945 88026

ORF 100431 100569

ORF 103282 103328

ORF 105787 105985

ORF 108936 110578

Allele FN:1394592CB1 44. 653 653 OG source isSNP SNP00124074 consequence FN: 1394592CB1.1 446 3'

Allele FN: 1394592CB1 445 749 749 A>G source isSNP SNP00124075 consequence FN: 1394592CB1.1 446 3' TABLE 1 (Cont.)

Allele FN: 1394592CB1 445 856 856 A>G source isSNP SNP00053065 consequence FN: 1394592CB1.1 446 3'

Allele FN: 1394592CB1 445 884 884 A>G source isSNP SNP00066145 consequence FN: 1394592CB1.1 446 3'

Allele FN: 1394592CB1 445 1048 1048 A>G source isSNP SNP00066146 consequence FN: 1394592CB1.1 446 3'

Allele FN: 1394592CB1 445 1214 1214 A>G source isSNP SNP00029910 consequence FN: 1394592CB1.1 446

GIF SDC4-cdna-fwd.gif Link : FL_1250708_link_genomic

Subsequence OB:HS453C12 1 147620 #271

Subsequenc :e GB:HS453C12_1394592CD1 87967 109084 #272

Subsequenc :e GB:HS453C12_2027624CD1 20194 10528 #273

Subsequence FL_1394592_mrna_build.1 87945 110578 #274

Subsequenc :e FL_2027624_mrna_build.l 20197 6152 #275

Subsequence OA21_cds.l 20194 17050 #276

CDS GB:HS453C12_1394592CD1 594 bp 5 exons #272 exon 87967 88026 exon 100431 100569 exon 103282 103328 exon 105787 105985 exon 108936 109084 mRNA FL_1394592_mrna_build.l 2110 bp 5 exons #274 exon 87945 88026 exon 100431 100569 exon 103282 103328 exon 105787 105985 exon 108936 110578

Allele GB:HS453C12 271 90320 90320 A>G source isSNP SNP00026142 consequence GB:HS453C12_1394592CD1 272 Intron

Allele GB:HS453C12 271 90420 90420 OG source isSNP SNP00026143 consequence GB:HS453C12_1394592CD1 272 Intron

Allele GB:HS453C12 271 96768 96768 A>G source dbSNP gnl|dbSNP|ss736312. _allel. consequence GB:HS453C12_1394592CD1 272 Intron

Allele GB:HS453C12 271 109121 109121 OG source isSNP SNP00124074 consequence GB:HS453C12_1394592CD1 272 3'

Allele GB:HS453C12 271 109217 109217 A>G source isSNP SNP00124075 consequence GB:HS453C12_1394592CD1 272 3'

Allele GB:HS453C12 271 109324 109324 A>G source isSNP SNP00053065 consequence GB:HS453C12_1394592CD1 272 3'

Allele GB:HS453C12 271 109352 109352 A>G source isSNP SNP00066145 consequence GB:HS453C12_1394592CD1 272 3'

Allele GB:HS453C12 271 109516 109516 A>G source isSNP SNP00066146 TABLE 1 (Cont.) consequence GB:HS453C12_1394592CD1 272 3' Allele GB:HS453C12 271 109682 109682 A>G source isSNP SNP00029910 consequence GB:HS453C12_1394592CD1 272 GIF SDC4-genomic-fwd.gif

SEDL

Full name : sedlin

Link : SEDL_link_cdna

Subsequence GB:NM_014563_1 1 2816 #447 CDS GB:NM_014563_1.1 423 bp #448

ORF 230 652 Allele GB:NM_014563_1 447 991 991 OT source dbSNP gnl | dbSNP] ss380525_allele source dbSNP gnl j dbSNPJ ss531221_allele consequence GB:NM_014563_1.1 448 3' Allele GB:NM_014563_1 447 2026 2026 A>G source dbSNP gnl | dbSNP| ss637643_allele source dbSNP gnl j dbSNpj ss869682_allele source dbSNP gnl j dbSNP| ssl272499_allele source dbSNP gnl j dbSNpj ss232503_allele source dbSNP gnl j dbSNpj ss459122_allele consequence GB:NM_014563_1.1 448 3' Allele GB:NM_014563_1 447 2391 2391 OG source isSNP SNP00010387 consequence GB:NM_014563_1.1 448 3' GIF SEDL-cdna-fwd.gif

SKI

Full name : v-ski avian sarcoma viral oncogene homolog

Link : SKI_link_cdna

Subsequence GB:NM_003036 1 3511 #449 CDS GB:NM_003036.1 2187 bp #450

ORF 73 2259 Allele GB:NM_003036 449 528 528 A>G source isSNP SNP00068450 consequence GB:NM_003036.1 4 45500 S Siilleenntt 1 15522--115522 R Allele GB:NM_003036 449 1146 1146 A>G source isSNP SNP00068451 consequence GB :NM_003036.1 450 Silent 358-358 Allele GB:NM_003036 449 3482 3482 OG source isSNP SNP00068452 consequence GB:NM_003036.1 450 GIF SKI-cdna-fwd.gif

SOD2

Full name : superoxide dismutase 2, mitochondrial

Link : SOD2_link_cdna

Subsequence EM:HSSOD 1 1026 #451 TABLE 1 (Cont.)

Allele EM:HSSOD 451 243 243 A>G source isSNP SNP00021476 Link : SOD2_link_genomic

Subsequence EM:S77127 101 12957 #452

Subsequence SOD2_link_cds .1 957 11597 #453

Subsequence SOD2_mrna_build.1 953 11950 #454 mRNA SOD2 _mrna_build.l 1026 bp 5 exons #454 exon 953 979 exon 1260 1462 exon 5859 5975 exon 9061 9240 exon 11452 11950

CDS SOD2 link cds.l 669 bp 5 exons #453 exon 957 979 exon 1260 1462 exon 5859 5975 exon 9061 9240 exon 11452 11597 Allele EM:S77127 452 1183 1183 A>G source isSNP SNP00003080 source wetSNP EM:S77127.vll83.C>T consequence SOD2_link_cds.1 453 Missense 16-16 A>V

Allele ΞM:S77127 452 1456 1456 OT source wetSNP EM:S77127.vl456.A>C consequence SOD2_link_cds.l 453 Intron

Allele EM:S77127 452 1734 1734 A>G source isSNP SNP00107369 consequence SOD2 link cds.l ^' 453 Intron

GIF SOD2-genomic-fwd.gif

SOD3

Full name : superoxide dismutase 3, extracellular

Link : SOD3_link_cdna

Subsequence GB:SOD3 1 1984 #455 CDS GB:SOD3.1 723 bp #456

ORF 664 1386

Allele GB : SOD3 455 835 835 A>G source isSNP SNP00033027 consequence GB:SOD3.1 456 Missense 58-58 T>A

Allele GB: SOD3 455 874 874 A>G source isSNP SNP00062433 consequence GB:S0D3.1 456 Silent 71-71 L

Allele GB: SOD3 455 1469 1469 A>G source isSNP SNP00067750 consequence GB-.SOD3.1 456 3'

Allele GB : SOD3 455 1496 1496 A>G source isSNP SNP00007500 consequence GB:SOD3.1 456 3'

Allele GB: SOD3 455 1817 1817 OT source isSNP SNP00104042 consequence GB:SOD3.1 456 3'

Allele GB : SOD3 455 1826 1826 A>G source isSNP SNP00031110 TABLE 1 (Cont.) consequence GB:SOD3.1 456 3 '

Allele GB : SOD3 455 1932 1932 A>G isSNP SNP00050239 consequence GB:SOD3.1 456 GIF SOD3-cdna-fwd . gif Link : FL_1534327_link_genomic

Subsequence GB : HSU10116 1 10079 #457

Subsequence GB : HSU10116_1534327CD1 5085 5807 #458

Subsequence FL_1534327_mrna_build . 1 1130 6405 #459 mRNA FL_1534327_mrna__build.l 1427 bp 2 exons #459 exon 1130 1219 exon 5069 6405

CDS GB:HSU10116_1534327CD1 723 bp 1 exon #458 exon 5085 5807

Allele GB:HSU10116 457 5256 5256 A>G source isSNP SNP00033027 consequence GB:HSU10116_1534327CD1 458 Missense 58-58 T>A

Allele GB:HSU10116 457 5295 5295 A>G source isSNP SNP00062433 consequence GB:HSU10116_1534327CD1 458 Silent 71-71 L

Allele GB:HSU10116 457 5890 5890 A>G source isSNP SNP00067750 consequence GB:HSU10116_1534327CD1 458

Allele GB:HSU10116 457 5917 5917 A>G source isSNP SNP00007500 consequence GB:HSU10116_1534327CD1 458

Allele GB:HSU10116 457 6238 6238 G>T source isSNP SNP00104042 consequence GB:HSU10116_1534327CD1 458

Allele GB:HSU10116 457 6247 6247 A>G source isSNP SNP00031110 consequence GB:HSU10116_1534327CD1 458

Allele GB:HSU10116 457 6353 6353 A>G source isSNP SNP00050239 consequence GB:HSU10116_1534327CD1 458

GIF SOD3-genomic-fwd.gif

SOX9

Full name : SOX9

Link : S0X9_link_cdna

Subsequence GB :HSSOX9MRN 3923 #460 CDS GB:HSSOX9MRN.2 1530 bp #461

ORF 360 1889 Allele GB:HSSOX9MRN 460 866 866 A>G source isSNP SNP00092616 consequence GB :HSSOX9MRN.2 461 Silent 169-169 H

Allele GB:HSSOX9MRN 460 1571 1571 A>G source isSNP SNP00108001 consequence GB :HSSOX9MRN.2 461 Silent 404-404

Allele GB:HSSOX9MRN 460 1912 1912 G>T source isSNP SNP00055269 consequence GB:HSSOX9MRN.2 461 3'

Allele GB:HSSOX9MRN 460 2374 2374 A>G TABLE 1 (Cont.) source isSNP SNP00041454 consequence GB:HSSOX9MRN.2 461 3' Allele GB:HSSOX9MRN 460 3224 3224 OG source isSNP SNP00061027 consequence GB:HSSOX9MRN.2 461 3' Allele GB:HSSOX9MRN 460 3470 3470 A>G source isSNP SNP00055270 consequence GB :HSSOX9MRN.2 461 GIF SOX9-cdna-fwd.gif Link : FL_5425567_link_genomic

Subsequence GB:AC007461_8_5425567CD1 63884 60889 #462 Subsequence GB:AC007461_8 1 180385 #463 Subsequence SOX9_mrna_build.1 64243 58856 #464

CDS GB:AC007461_8_5425567CD1 1530 bp 3 exons #462 exon 63884 63454 exon 62557 62304 exon 61733 60889 mRNA SOX9_mrna_build.1 3922 bp 3 exons #464 exon 64243 63454 exon 62557 62304 exon 61733 58856

Allele GB:AC007461 8 463 59309 59309 A>G source isSNP SNP00055270 consequence GB:AC007461_8_5425567CD1 462

Allele GB:AC007461_8 463 59555 59555 OG source isSNP SNP00061027 consequence GB:AC007461_8_5425567CD1 462

Allele GB:AC007461_8 463 60078 60078 A>G source isSNP SNP00010889 consequence GB:AC007461_8_5425567CDl 462

Allele GB:AC007461_8 ^°463 60404 60404 A>G source isSNP SNP00041454 consequence GB:AC007461_8_5425567CD1 462

Allele GB:AC007461_8 463 60866 60866 G>T source isSNP SNP00055269 consequence GB:AC007461_8_5425567CDl 462

Allele GB:AC007461_8 463 61207 61207 A>G source isSNP SNP00108001 consequence GB:AC007461_8_5425567CDl 462 Silent

404-404 P Allele GB:AC007461_8 463 62482 62482 A>G source isSNP SNP00092616 source wetSNP GB:AC007461_8.v62482.G>A consequence GB:AC007461_8_5425567CD1 462 Silent

169-169 H

GIF SOX9-genomic-rev.gif

STATI2

Full name : STAT-induced STAT inhibitor-2

Link : FL_2787140_link_cdna

Subsequence FN: 2787140CB1 1 2587 #465

CDS FN:2787140CB1.1 927 bp #466

ORF 98 1024 TABLE 1 (Cont.)

Allele FN: 2787140CB1 465 1325 1325 A>G source isSNP SNP00041483 consequence FN: 2787140CB1.1 466 3'

Allele FN: 2787140CB1 465 1442 1442 G>T source isSNP SNP00106962 consequence FN: 2787140CB1.1 466 3'

Allele FN: 2787140CB1 465 1470 1470 A>G source isSNP SNP00041484 consequence FN: 2787140CB1.1 466 3'

Allele FN: 2787140CB1 465 1974 1974 A>G source isSNP SNP00106963 consequence FN: 2787140CB1.1 466

GIF STATI2-cdna-fwd.gif Link : FL_1405668_link_genomic

Subsequence GB :AC012085_1 1 177866 #467

Subsequence FL_2787140_mrna_build.1 42013 47745 #468 mRNA FL_2787140_mrna_b ild.l 2580 bp exons #46. exon 42013 42225 exon 43694 44045 exon 45731 47745

Allele GB:AC012085_1 467 44268 44268 A>G source isSNP SNP00070304

Allele GB:AC012085. 467 46492 46492 A>G source isSNP SNP00041483

Allele GB:AC012085_. 467 46609 46609 G>T source isSNP SNP00106962

Allele GB:AC012085_. 467 46637 46637 A>G source isSNP SNP00041484

Allele GB:AC012085_1 467 47141 47141 A>G source isSNP SNP00106963 GIF STATI2-genomic-fwd.gif

THBS1

Full name : thrombospondin 1

Link : THBSl_link_cdna

Subsequence GB :HSTS 5722 #469 CDS GB:HSTS.l 3513 bp #470

ORF 112 3624

Allele GB:HSTS 469 1239 1239 A>G source isSNP SNP00046537 consequence GB:HSTS.l 470 Silent 376-376 D

Allele GB :HSTS 469 2210 2210 A>G source isSNP SNP00046539 consequence GB:HSTS.l 470 Missense 700-700 N>S

Allele GB:HSTS 469 2979 2979 A>G source isSNP SNP00061983 consequence GB:HSTS.l 470 Silent 956-956 D

Allele GB :HSTS 469 3680 3680 G>T source isSNP SNP00108514 consequence GB:HSTS.l 470 3'

Allele GB:HSTS 469 3703 3703 A>G source isSNP SNP00013197 consequence GB:HSTS.l 470 TABLE 1 (Cont.)

Allele GB:HSTS 469 3905 3905 A>G source isSNP SNP00093327 consequence GB:HSTS.l 470 3'

Allele GB:HSTS 469 5259 5259 A>G source isSNP SNP00105437 consequence GB:HSTS.l 470 3'

GIF THBSl-cdna-fwd.gif

TIMP1

Full name : Tissue Inhibitor of Metalloproteinase

Link : TIMPl_link_cdna

Subsequence FN:411388CB1 1 853 #471 CDS FN.411388CB1.1 621 bp #472

ORF 122 742 Allele FN:411388CB1 471 365 365 OG source isSNP SNP00115174 consequence FN: 411388CB1.1 472 Missense 82-82 R>G GIF TIMPl-cdna-fwd.gif Link : FL_3013907_link_genomic

Subsequence OB:HS230G1 1 125515 #473 Subsequence GB:HS230G1_411388CD1 20559 17287 #474 Subsequence TIMPl_mrna_build.1 21613 17186 #475 mRNA TIMPl_mrna_buiId.1 843 bp 6 exons #475 exon 21613 21501 eexxoonn 20567 2200443399 eexxoonn 19039 1188996600 eexxoonn 18770 1188664444 eexxoonn 18432 1188330088 eexxoonn 17454 1177118866

CDS GB:HS230G1_411388CD1 621 bp 5 exons #474 exon 20559 20439 exon 19039 18960 exon 18770 18644 exon 18432 18308 exon 17454 17287

Allele GB:HS230G1 4 473 17434 17434 A>G source wwetSNP GB:HS230Gl.vl7434.G>A consequence G G!B :HS230G1_411388CD1 474 Silent 158-158

I Allele GB:HS230G1 473 17550 17550 A>G source isSNP SNP00099224 consequence GB:HS230G1_411388CD1 474 Intron

Allele GB:HS230G1 473 18046 18046 A>G source isSNP SNP00099223 consequence GB:HS230G1_411388CD1 474 Intron

Allele GB:HS230Gl 473 18088 18088 A>G source isSNP SNP00030937 consequence GB:HS230G1_411388CD1 474 Intron

Allele GB:HS230G1 473 18389 18389 A>G source wetSNP GB:HS230G1.vl8389.A>G consequence GB:HS230G1_411388CD1 474 Silent 124-124

F Allele GB : HS230G1 473 18495 18495 OG TABLE 1 (Cont.) source isSNP SNP00099222 source wetSNP GB:HS230G1.vl8495.OG consequence GB:HS230G1_411388CD1 474 Intron

Allele GB:HS230Gl 473 18711 18711 A>G source wetSNP GB:HS230G1.vl8711.G>A consequence GB:HS230G1_411388CD1 474 Silent 87-87 P

Allele GB:HS230G1 473 18728 18728 OG source isSNP SNP00115174 consequence GB:HS230G1_411388CD1 474 Missense 82-82 R>G GIF TIMPl-genomic-rev.gif

TIMP2

Full name : Tissue Inhibitor of Metalloproteinase-2.

Link : TIMP2_link_genomic

Subsequence TIMP2. _cds .1 822 3126 #476

Subsequence GB:S68860_1 1 970 #477

Subsequence GB:U44382_1 1071 1320 #478

Subsequence GB:U44383_1 1421 1644 #479

Subsequence GB:U44384_1 1745 2283 #480

Subsequence GB:U44385_1 2384 3750 #481

Subsequence TIMP2. _mrna_build.1 810 3251 #<

CDS TIMP2_ .eds .1 663 bp 5 exons #476 exon 822 951 exon 1125 1225 exon 1504 1612 exon 1939 2063 exon 2929 3126 mRNA TIMP2. _mrna_build.1 800 bp 5 exons #482 exon 810 951 exon 1125 1225 exon 1504 1612 exon 1939 2063 exon 2929 3251

Allele GB:U44383_1 479 155 155 A>G source wetSNP GB:U44383_1, .vl55.G>A consequence TIMP2_ .eds .1 476 Silent 1 1(01-101

GIF TIMP2-genomic-fwd.gif

TNA

Full name : tetranectin

Link : TNA_link_cdna

Subsequence GB:NM_003278 874 #483 CDS GB:NM_003278.1 609 bp #484

ORF 94 702 Allele GB:NM_003278 483 409 409 A>G source isSNP SNP00007942 consequence GB:NM_003278.1 484 Missense 106-106 S>G

Allele GB:NM_003278 483 744 744 A>G source isSNP SNP00007943 consequence GB:NM_003278.1 484 GIF TNA-cdna-fwd.gif „ TABLE 1 (Cont.)

Link : TNA_link_genomic

Subsequence TNA_cds .1 254 1629 #485 Subsequence TNA_cds .2 254 1629 #486 Subsequence GB:X70910_ 1 570 #487 Subsequence GB:X70911_ 671 978 #488 Subsequence GB:X70912 1079 1805 #489 Subsequence TNA_mrna build.1 164 1776 #490 CDS TNA_cds.l 609 bp 3 exons #485 exon 254 362 exon 829 927 exon 1229 1629 CDS TNA_cds.2 510 bp 2 exons #486 exon 254 362 exon 1229 1629 mRNA TNA_mrna_bui1d 846 bp 3 exons #490 exon 164 362 exon 829 927 exon 1229 1776 Allele GB:X70912_1 489 258 258 A>G source isSNP SNP00007942 consequence TNA_cds.l 485 Missense 106-106 S>G consequence TNA_cds.2 486 Missense 73-73 S>G

Allele GB:X70912_1 489 593 593 A>G source isSNP SNP00007943 consequence TNA_cds.l 485 3' consequence TNA_cds.2 486 3'

GIF TNA-genomic-fwd.gif

TNFAIP6

Full name : tumor necrosis factor, alpha-induced protein 6

Link : TNFAIP6_link_cdna

Subsequence GB:NM_007115_1 1 1414 #491 CDS GB:NM_007115_1.1 834 bp #492

ORF 69 902 Allele GB:NM_007115_1 491 499 499 A>G source isSNP SNP00040822 consequence GB:NM_007115_1.1 492 Missense 144-144 R>Q

Allele GB:NM_007115_1 491 1143 1143 OG source isSNP SNP00040823 consequence GB:NM_007115_1.1 492 GIF TNFAIP6-cdna-fwd.gif Link : FL_1000909_link_genomic

Subsequence GB:AC009311_1_1919, .8CD1 132384 154250 #493 Subsequence GB:AC009311_1 1 160198 #494 Subsequence TNFAIP6_mrna_build 1 132314 154760 #495 mRNA TNFAIP6_mrna_build.l 1414 bp 6 exons #495 exon 132314 132477 exon 138660 138797 exon 140773 140934 exon 144737 144965 exon 148266 148306 exon 154081 154760

CDS GB:AC009311_1_191918CD1 834 bp exons TABLE 1 (Cont.) exon 132384 132477 exon 138660 138797 exon 140773 140934 exon 144737 144965 exon 148266 148306 exon 154081 154250

Allele GB.AC009311. _.1 494 140934 140934 A>G source wetSNP GB:AC009311..l.vl40934.G>A consequence GB:AC009311_1_191918CD1 493 Missense 132-132

A>T

Allele GB.AC009311. .1 494 140942 140942 A>T source wetSNP GB:AC009311_ l.vl40942.A>T consequence GB:AC009311_._1_191918CD1 493 Intron

Allele GB.AC009311.1 494 144773 144773 A>G source isSNP SNP00040822 source wetSNP GB:AC009311_ .l.vl44773.A>G consequence GB:AC009311_1_191918CD1 493 Missense 144-144

Q>R

Allele GB:AC009311. .1 494 148030 148030 A>G source dbSNP gnl|dbSNP|ss645109. _allele consequence GB:AC009311_1_191918CD1 493 Intron

Allele GB.AC009311 .1 494 148229 148229 A>G source wetSNP GB:AC009311_ ,l.vl48229.T>C consequence GB:AC009311_1_191918CD1 493 Intron

Allele GB:AC009311. _.1 494 148245 148245 A>G source wetSNP GB:AC009311_ ,l.vl48245.T>C consequence GB:AC009311_1_191918CD1 493 Intron

Allele GB:AC009311. .1 494 154493 154493 OG source isSNP SNP00040823 consequence GB:AC009311 1 191918CD1 493 3'

GIF TNFAIP6-genomic-fwd.gif

TNFRSF11B

Full name : TNFRSF11B Link : TNFRSFllB_link_cdna Subsequence GB:AB002146 1206 #496

CDS GB:AB002146.1 1206 bp #497

ORF 1 1206 Allele GB:AB002146 496 768 768 A>G source isSNP SNP00028816 consequence GB:AB002146.1 497 Silent 256-256 GIF TNFRSFllB-cdna-fwd.gif Link : TNFRSFllB_link_genomic

Subsequence TNFRSFllB_cds .1 125 9057 #498 Subsequence GB:E15270_1 1 9898 #499

CDS TNFRSFllB_cds .1 1176 bp 4 exons #498 exon 130 499 exon 4504 4695 exon 6716 6940 exon 8669 9057 Allele GB:E15270_1 499 503 503 A>G source wetSNP GB:E15270_1. V503.OT consequence TNFRSF11B cds.l 498 Intron TABLE 1 (Cont.)

Allele GB:E15270_1 499 4499 4499 A>G source wetSNP GB:E15270_1 ,v4499.C>T consequence TNFRSFllB_cds.l 498 Intron

Allele GB:E15270_1 499 4661 4661 A>G source wetSNP GB:E15270_1 ,v4661.C>T consequence TNFRSFllB_cds .1 498 Silent 176-176

Allele GB:E15270_1 499 4749 4752 TCTOTG source wetSNP GB : E15270_l .v4749 .TCTG>TG consequence TNFRSFllB_cds.l 498 Intron

Allele GB:E15270_1 499 6599 6599 A>G source wetSNP GB:E15270_l.v6599.G>A consequence TNFRSFllB_cds .1 498 Intron

Allele GB:E15270_1 499 6837 6837 A>G source wetSNP GB:E1! 270_l.v6837.G>A consequence TNFRSFllB_cds.l 498 Silent 228-228

Allele GB:E15270_1 499 6891 6891 A>G source isSNP SNP00028816 consequence TNFRSFllB_cds.l 498 Silent 246-246

GIF TNFRSFllB-genomic- fwd.gif

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Claims

What is claimed is:

1. A method of deterrnining susceptibility of an individual to joint space narrowing and/or osteophyte development and/or joint pain comprising identifying whether the individual has at least one polymorphism in a polynucleotide encoding at least one of the proteins listed in Table 1.

2. The method of claim 1, wherein said proteins listed in Table 1 are selected from the group consisting of bone morphogenic protein 2 (BMP2), cartilage intermediate layer protein (CILP), cartilage oligomeric matrix protein (COMP), tissue inhibitor of metalloproteinase 1 (TTMP1), tetranectin (TNA), matrix metalloproteinase 3 (MMP3), and prostaglandin-endoperoxide synthase 2 (PTGS2).

3. The method of claim 1, wherein the joint space narrowing and/or osteophyte development and/or joint pain is associated with a disease.

4. The method of claim 3 wherein the disease is osteoarthritis.

5. The method of claim 1 where at least one of the polymorphisms is selected from the polymorphisms listed in Table 1.

6. The method of claim 1 comprising contacting a sample from the individual with a specific binding agent for the polymorphism and deteπmning whether the agent binds to the polymorphism.

7. The method of claim 1 where the polymorphism in the polynucleotide is determined for more than one allele of the individual.

8. A method for modulating the susceptibility of an individual to joint space narrowing and/or osteophyte development and/or joint pain, comprising identifying the individual by the method of claim 1 and adn inistering to the individual a composition comprising an effective amount of an agent which modulates said susceptibility.

9. The method of claim 8, wherein the joint space narrowing and/or osteophyte development and/or joint pain is associated with a disease.

10. The method of claim 9 wherein the disease is osteoarthritis.

11. A polynucleotide encoding a protein listed in Table 1 having at least one polymorphism in the polynucleotide selected from the group of polymorphisms listed in Table 1 for the polynucleotide.

12. A fragment of a polynucleotide encoding a protein selected from Table 1 having at least one polymorphism in the fragment selected from the group of polymorphisms listed in Table 1.

13. A fragment of claim 12 having a length of 8 to 100 nucleotides.

14. A fragment of claim 12 having a length of 8 to 30 nucleotides.

15. A fragment of claim 12 having a length of 9 to 15 nucleotides.

16. A method of identifying an agent for modulating susceptibility of an individual to joint space narrowing and/or osteophyte development and/or joint pain comprising: a) contacting a test agent with a polypeptide or a polynucleotide encoding the polypeptide selected from the list of Table 1 having at least one of the polymorphisms selected from the list of Table 1, b) deteπrjining whether the agent is capable of binding to the polypeptide or polynucleotide encoding the polypeptide, and c) deterrjjj_ing whether the activity or expression of the polypeptide or polynucleotide encoding the polypeptide is modulated.

17. A method of formulating a composition comprising a) identifying an agent for modulating the susceptibility of an individual to joint space narrowing and/or osteophyte development and/or joint pain by the method of claim 16, and b) formulating the agent with a carrier or diluent.

18. An agent identified by the method of claim 16.

19. A composition for modulating the susceptibility of an individual to joint space narrowing and/or osteophyte development and/or joint pain comprising an agent according to claim 18 and a carrier.

20. A method comprising using an agent of claim 18 in the manufacture of a medicament for modulating susceptibility to joint space narrowing and/or osteophyte development and/or joint pain.

21. A probe, primer or antibody which is capable of selectively detecting a polymorphism listed in Table 1 which is associated with susceptibility to joint space narrowing and/or osteophyte development and/or joint pain.

22. A vector comprising the polynucleotide of claim 11.

23. A host cell line comprising the vector of claim 22.

24. A nonhuman animal which is transgenic for the polynucleotide of claim 11.

25. A cell line comprising the polynucleotide of claim 11.

26. A method of using a cell line of claim 25 to screen for an agent for diagnosis of an individual having susceptibility to joint space nanowing and/or osteophyte development and/or joint pain.

27. A method of using a nonhuman animal of claim 24 to screen for an agent for diagnosis of an individual having susceptibility to joint space narrowing and/or osteophyte development and/or joint pain.

28. A kit for diagnosis of an individual having susceptibility to joint space narrowing and/or osteophyte development and/or joint pain comprising an agent for detection of the polynucleotide of claim 11.

29. The kit of claim 28 further comprising instruction for use of said agent for detection of said polynucleotide.

30. A kit for diagnosis of an individual having susceptibility to joint space nanowing and/or osteophyte development and/or joint pain comprising an agent for detection of the fragment of a polynucleotide of claim 12.

31. The kit of claim 30 further comprising instructions for use of said agent for detection of said fragment.

32. A kit for diagnosis of an individual having susceptibility to joint space nanowing and/or osteophyte development and/or joint pain comprising the probe, primer or antibody of claim 21.

33. The kit of claim 32 further comprising instructions for use of said probe, primer or antibody.